Compositions and methods for minimizing nornicotine synthesis in tobacco

ABSTRACT

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in tobacco plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for a root-specific nicotine demethylases, CYP82E10, and variants thereof, that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Compositions of the invention also include tobacco plants, or plant parts thereof, comprising a mutation in a gene encoding a CYP82E10 nicotine demethylase, wherein the mutation results in reduced expression or function of the CYP82E10 nicotine demethylase. Seed of these tobacco plants, or progeny thereof, and tobacco products prepared from the tobacco plants of the invention, or from plant parts or progeny thereof, are also provided. Methods for reducing the level of nornicotine, or reducing the rate of conversion of nicotine to nornicotine, in a tobacco plant, or plant part thereof are also provided. The methods comprise introducing into the genome of a tobacco plant a mutation within at least one allele of each of at least three nicotine demethylase genes, wherein the mutation reduces expression of the nicotine demethylase gene, and wherein a first of these nicotine demethylase genes encodes a root-specific nicotine demethylase involved in the metabolic conversion of nicotine to nornicotine in a tobacco plant or a plant part thereof. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products.

INCORPORATION OF SEQUENCE LISTING

An official copy of the Sequence Listing is submitted electronically viaEFS-Web as an ASCII formatted Sequence Listing with a file named“13521766SeqListReplacement.txt,” created on Jan. 20, 2015, having asize of 150 KB and is filed concurrently with the substitutespecification. The Sequence Listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to compositions and methods for minimizingnornicotine synthesis, and hence its metabolite N′-nitrosonornicotine,in tobacco plants and plant parts thereof, particularly compositions andmethods for inhibiting expression or function of a root-specificnicotine demethylase in combination with a green leaf and asenescence-induced nicotine demethylase.

BACKGROUND OF THE INVENTION

The predominant alkaloid found in commercial tobacco varieties isnicotine, typically accounting for 90-95% of the total alkaloid pool.The remaining alkaloid fraction is comprised primarily of threeadditional pyridine alkaloids: nornicotine, anabasine, and anatabine.Nornicotine is generated directly from nicotine through the activity ofthe enzyme nicotine N-demethylase. Nornicotine usually represents lessthan 5% of the total pyridine alkaloid pool, but through a processtermed “conversion,” tobacco plants that initially produce very lowamounts of nornicotine give rise to progeny that metabolically “convert”a large percentage of leaf nicotine to nornicotine. In tobacco plantsthat have genetically converted (termed “converters”), the greatmajority of nornicotine production occurs during the senescence andcuring of the mature leaf (Wernsman and Matzinger (1968) Tob. Sci.12:226-228). Burley tobaccos are particularly prone to geneticconversion, with rates as high as 20% per generation observed in somecultivars.

During the curing and processing of the tobacco leaf, a portion of thenornicotine is metabolized to the compound N-nitrosonornicotine (NNN), atobacco-specific nitrosamine (TSNA) that has been asserted to becarcinogenic in laboratory animals (Hecht and Hoffmann (1990) CancerSurveys 8:273-294; Hoffmann et al. (1994) J. Toxicol. Environ. Health41:1-52; Hecht (1998) Chem. Res. Toxicol. 11:559-603). In flue-curedtobaccos, TSNAs are found to be predominantly formed through thereaction of alkaloids with the minute amounts of nitrogen oxides presentin combustion gases formed by the direct-fired heating systems found intraditional curing barns (Peele and Gentry (1999) “Formation ofTobacco-specific Nitrosamines in Flue-cured Tobacco,” CORESTA Meeting,Agro-Phyto Groups, Suzhou, China). Retrofitting these curing barns withheat-exchangers virtually eliminated the mixing of combustion gases withthe curing air and dramatically reduced the formation of TSNAs intobaccos cured in this manner (Boyette and Hamm (2001) Rec. Adv. Tob.Sci. 27:17-22.). In contrast, in the air-cured Burley tobaccos, TSNAformation proceeds primarily through reaction of tobacco alkaloids withnitrite, a process catalyzed by leaf-borne microbes (Bush et al. (2001)Rec. Adv. Tob. Sci. 27:23-46). Thus far, attempts to reduce TSNAsthrough modification of curing conditions while maintaining acceptablequality standards have not proven to be successful for the air-curedtobaccos.

Aside from serving as a precursor for NNN, recent studies suggest thatthe nornicotine found in tobacco products may have additionalundesirable health consequences. Dickerson and Janda (2002) Proc. Natl.Acad. Sci. USA 99: 15084-15088 demonstrated that nornicotine causesaberrant protein glycation within the cell. Concentrations ofnornicotine-modified proteins were found to be much higher in the plasmaof smokers compared to nonsmokers. This same study also showed thatnornicotine can covalently modify commonly prescribed steroid drugs suchas prednisone. Such modifications have the potential of altering boththe efficacy and toxicity of these drugs. Furthermore, studies have beenreported linking the nornicotine found in tobacco products withage-related macular degeneration, birth defects, and periodontal disease(Brogan et al. (2005) Proc. Natl. Acad. Sci. USA 102: 10433-10438; Katzet al. (2005) J. Periodontol. 76: 1171-1174).

In Burley tobaccos, a positive correlation has been found between thenornicotine content of the leaf and the amount of NNN that accumulatesin the cured product (Bush et al. (2001) Rec. Adv. Tob. Sci, 27:23-46;Shi et al. (2000) Tob. Chem. Res. Conf. 54:Abstract 27). Therefore,strategies that could effectively reduce the nornicotine content of theleaf would not only help ameliorate the potential negative healthconsequences of the nornicotine per se as described above, but shouldalso concomitantly reduce NNN levels. This correlation was furthersolidified in the recent study by Lewis et al. (2008) Plant Biotech. J.6: 346-354 who demonstrated that lowering nornicotine levels using anRNAi transgene construct directed against the CYP82E4v2 gene, whichencodes a senescence-induced nicotine demethylase, lead to concomitantreductions in the NNN content of the cured leaf. Although this studydemonstrated that transgenic technologies can be used to greatly reducethe nornicotine and NNN content of tobacco, a combination of publicperception and intellectual property issues make it very difficult forcommercialization of products derived from transgenic plants.

Therefore a great need exists for a means to effectively minimizenornicotine accumulation in tobacco that does not rely on the use oftransgenics.

SUMMARY OF THE INVENTION

Compositions and methods for minimizing the nornicotine content intobacco plants and plant parts thereof are provided. Compositionsinclude an isolated root-specific cytochrome P450 polynucleotidedesignated the CYP82E10 polynucleotide, as set forth in SEQ ID NO:1, andCYP82E10 nicotine demethylase polypeptide encoded thereby, as set forthin SEQ ID NO:2, and variants and fragments thereof, including, but notlimited to, polypeptides comprising the sequence set forth in SEQ IDNO:5, 6, 7, 8, 9, 10, 11, 12, or 13, as well as polynucleotides encodingthe polypeptide set forth in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, or 13.The CYP82E10 polypeptide of the invention is a nicotine demethylase thatis involved in the metabolic conversion of nicotine to nornicotine inthe roots of tobacco plants. Isolated polynucleotides of the inventionalso include a polynucleotide comprising the sequence set forth in SEQID NO:3 or 4, and variants and fragments thereof. Compositions of theinvention also include tobacco plants, or plant parts thereof,comprising a mutation in a gene encoding a CYP82E10 nicotinedemethylase, wherein the mutation results in reduced expression orfunction of the CYP82E10 nicotine demethylase. In some embodiments, thetobacco plants of the invention further comprise a mutation in a geneencoding a CYP82E4 nicotine demethylase and/or a mutation in a geneencoding a CYP82E5 nicotine demethylase, wherein the mutation withinthese genes results in reduced expression or function of the CYP82E4 orCYP82E5 nicotine demethylase. Seed of these tobacco plants, or progenythereof, and tobacco products prepared from the tobacco plants of theinvention, or from plant parts or progeny thereof, are also provided.

Methods for reducing the level of nornicotine, or reducing the rate ofconversion of nicotine to nornicotine, in a tobacco plant, or plant partthereof are also provided. The methods comprise introducing into thegenome of a tobacco plant a mutation within at least one allele of eachof at least three nicotine demethylase genes, wherein the mutationreduces expression of the nicotine demethylase gene, and wherein a firstof these nicotine demethylase genes encodes a root-specific nicotinedemethylase involved in the metabolic conversion of nicotine tonornicotine in a tobacco plant or a plant part thereof. In someembodiments, the root-specific nicotine demethylase is CYP82E10 orvariant thereof. In other embodiments, these methods compriseintroducing into the genome of a tobacco plant a mutation within atleast one allele of a nicotine demethylase gene encoding CYP82E10 orvariant thereof, and a mutation within at least one allele of a nicotinedemethylase encoding CYP82E4 or variant thereof, and/or a nicotinedemetylase encoding CYP82E5 or variant thereof. Methods for identifyinga tobacco plant with low levels of nornicotine are also provided,wherein the plant or plant part thereof is screened for the presence ofa mutation in a gene encoding CYP82E10 or variant thereof, alone or incombination with screening for the presence of a mutation in a geneencoding CYP82E4 or variant thereof, and/or the presence of a mutationin a gene encoding CYP82E5 or variant thereof.

The following embodiments are encompassed by the present invention.

1. A tobacco plant, or plant part thereof, comprising a mutation in agene encoding a CYP82E10 nicotine demethylase, wherein said mutationresults in reduced expression or function of said CYP82E10 nicotinedemethylase.

2. The tobacco plant, or plant part thereof, according to embodiment 1,wherein said CYP82E10 nicotine demethylase is selected from the groupconsisting of the sequence set forth in SEQ ID NO:2, 5, 6, 7, 8, and 9.

3. The tobacco plant, or plant part thereof, according to embodiment 1or 2, wherein said mutation results in a modification of said CYP82E10nicotine demethylase occurring at a position selected from the groupconsisting of amino acid residues 79, 107, 381, 419, and any combinationthereof, wherein said numbering is according to SEQ ID NO:2.

4. The tobacco plant, or plant part thereof, according to embodiment 3,wherein said mutation is selected from the group consisting of:

-   -   a) a serine substitution for the glycine residue at position 79;    -   b) a serine substitution for the proline residue at position        107;    -   c) a serine substitution for the proline residue at position        381;    -   d) a serine substitution for the proline residue at position        419; and    -   e) any combination thereof.

5. The tobacco plant, or plant part thereof, according to any ofembodiments 1-4, further comprising a mutation in a gene encoding aCYP82E4 nicotine demethylase, wherein said mutation results in reducedexpression or function of said CYP82E4 nicotine demethylase.

6. The tobacco plant, or plant part thereof, according to embodiment 5,wherein said CYP82E4 nicotine demethylase is selected from the sequenceset forth in SEQ ID NO:14, 15, 16, 17, 18, 19, and 20.

7. The tobacco plant, or plant part thereof, according to embodiment 5or 6, wherein said mutation results in a modification of said CYP82E4nicotine demethylase occurring at a position selected from the groupconsisting of amino acid residues 329, 364, 376, 381, and 458, whereinsaid numbering is according to SEQ ID NO:14.

8. The tobacco plant, or plant part thereof, according to embodiment 7,wherein said mutation is selected from the group consisting of:

-   -   a) a stop codon substitution for the tryptophan residue at        position 329;    -   b) an asparagine substitution for the lysine residue at position        364;    -   c) a methionine substitution for the valine residue at position        376;    -   d) a serine substitution for the proline residue at position        381;    -   d) a serine substitution for the proline residue at position        458; and    -   e) any combination thereof.

9. The tobacco plant, or plant part thereof, according to any ofembodiments 1-8, further comprising a mutation in a gene encoding aCYP82E5 nicotine demethylase, wherein said mutation results in reducedexpression or function of said CYP82E5 nicotine demethylase.

10. The tobacco plant, or plant part thereof, according to embodiment 9,wherein said CYP82E5 nicotine demethylase is selected from the sequenceset forth in SEQ ID NO:26, 27, 28, 29, 30, 31, and 32.

11. The tobacco plant, or plant part thereof, according to embodiment 9or 10, wherein said mutation results in a modification of said CYP82E5nicotine demethylase occurring at a position selected from the groupconsisting of amino acid residues 422 and 449, wherein said numbering isaccording to SEQ ID NO:26.

12. The tobacco plant, or plant part thereof, according to embodiment11, wherein said mutation is selected from the group consisting of:

-   -   a) a stop codon substituted for the tryptophan residue at        position 422;    -   b) a leucine substituted for the proline residue at position        449; and    -   c) any combination thereof.

13. The tobacco plant, or plant part thereof, according to any ofembodiments 9-12, comprising a mutation in said CYP82E10 nicotinedemethylase gene and said CYP82E4 nicotine demethylase gene.

14. The tobacco plant, or plant part thereof, according to any ofembodiments 1-13, wherein said tobacco plant, or plant part thereof, ishomozygous for said mutation.

15. The tobacco plant, or plant part thereof, according to embodiment14, wherein said CYP82E10 nicotine demethylase comprises a mutation atposition 381, said CYP82E4 nicotine demethylase comprises a mutation atposition 329, and said CYP82E5 nicotine demethylase comprises a mutationat position 422, wherein said numbering is according to SEQ ID NO:2, 14,and 26, respectively.

16. The tobacco plant, or plant part thereof, according to embodiment15, wherein said mutation is selected from the group consisting of:

-   -   a) a serine substitution for the proline residue at position        381;    -   b) a stop codon substitution for the tryptophan residue at        position 329;    -   c) a stop codon substitution for the tryptophan residue at        position 422; and    -   d) any combination thereof.

17. The tobacco plant, or plant part thereof, according to any ofembodiments 13-16, wherein said plant or plant part thereof has lessthan 1.5% conversion of nicotine to nornicotine.

18. The tobacco plant, or plant part thereof, according to embodiment17, wherein said plant or plant part thereof has no more than 0.5%conversion of nicotine to nornicotine.

19. Seed of the tobacco plant according to any of embodiments 1-18, orprogeny thereof.

20. A tobacco product prepared from a tobacco plant, or plant part orprogeny thereof, according to any of embodiments 1-19.

21. A method for reducing a carcinogenic potential of a tobacco product,said method comprising preparing said tobacco product from a tobaccoplant, or plant part or progeny thereof, according to any of embodiments1-18.

22. A method for reducing the level of nornicotine, or reducing the rateof conversion of nicotine to nornicotine, in a tobacco plant, or a plantpart thereof, said method comprising introducing into the genome of saidplant a mutation within at least one allele of each of at least threenicotine demethylase genes, wherein said mutation reduces expression ofsaid nicotine demethylase gene, and wherein a first of said nicotinedemethylase genes encodes a root-specific nicotine demethylase involvedin the metabolic conversion of nicotine to nornicotine in a tobaccoplant or a plant part thereof.

23. The method of embodiment 22, wherein said root-specific nicotinedemethylase is a CYP82E10 nicotine demethylase comprising an amino acidsequence selected from the group consisting of:

-   -   a) the amino acid sequence set forth in SEQ ID NO:2, 5, 6, 7, 8,        9, or 10; and    -   b) an amino acid sequence having at least 98% sequence identity        to the amino acid sequence set forth in SEQ ID NO:2, 5, 6, 7, 8,        9, or 10.

24. The method of embodiment 23, wherein said amino acid sequence forsaid CYP82E10 nicotine demethylase has a substitution at an amino acidresidue in a position selected from the group consisting of residues 79,107, 381, 419, and any combination thereof, where the numbering isaccording to SEQ ID NO:2.

25. The method of embodiment 24, wherein said substitution at position79, 107, 381, or 419 is a serine residue.

26. The method of any one of embodiments 22-25, wherein a second of saidnicotine demethylase genes encodes a CYP82E4 nicotine demethylase.

27. The method of embodiment 26, wherein said CYP82E4 nicotinedemethylase comprises an amino acid sequence selected from the groupconsisting of:

-   -   a) the amino acid sequence set forth in SEQ ID NO:14, 15, 16,        17, 18, 19, 20, or 21; and    -   b) an amino acid sequence having at least 98% sequence identity        to the sequence set forth in SEQ ID NO:14, 15, 16, 17, 18, 19,        20, or 21.

28. The method of embodiment 27, wherein said amino acid sequence forsaid CYP82E4 nicotine demethylase has a substitution at an amino acidresidue in a position selected from the group consisting of residues329, 364, 381, 458, and any combination thereof, where the numbering isaccording to SEQ ID NO:14.

29. The method of embodiment 28, wherein said substitution at position329 is a stop codon, said substitution at position 364 is an asparagineresidue, said substitution at position 381 is a serine residue, saidsubstitution at position 458 is a serine residue, or any combinationthereof.

30. The method of any one of embodiments 22-29, wherein a third of saidnicotine demethylase genes encodes a CYP82E5 nicotine demethylase.

31. The method of embodiment 30, wherein said CYP82E5 nicotinedemethylase comprises an amino acid sequence selected from the groupconsisting of:

-   -   a) the amino acid sequence set forth in SEQ ID NO:26, 27, 28,        29, 30, 31, or 32; and    -   b) an amino acid sequence having at least 98% sequence identity        to the sequence set forth in SEQ ID NO: 26, 27, 28, 29, 30, 31,        or 32.

32. The method of embodiment 31, wherein said amino acid sequence forsaid CYP82E5 nicotine demethylase has a substitution at an amino acidresidue in a position selected from the group consisting of residues 422and 449, and any combination thereof, where the numbering is accordingto SEQ ID NO:26.

33. The method of embodiment 32, wherein said substitution at position422 is a stop codon, said substitution at position 449 is a leucineresidue, or any combination thereof.

34. The method of any one of embodiments 22-33, wherein said plant orplant part thereof is homozygous for said mutation.

35. The method of any one of embodiments 22-34, wherein said introducingcomprises a breeding protocol.

36. The method of any one of embodiments 22-35, wherein said plant is aBurley, Va., flue-cured, air-cured, fire-cured, Oriental, or a darktobacco plant.

37. The tobacco plant, or plant part thereof, according to any ofembodiments 1-18, wherein said tobacco plant is a Burley, Va.,flue-cured, air-cured, fire-cured, Oriental, or a dark tobacco plant.

38. A method for identifying a tobacco plant with low levels ofnornicotine, said method comprising screening a DNA sample from atobacco plant of interest for the presence of a mutation in SEQ ID NO:1or 3.

39. The method according to embodiment 38, wherein said tobacco plant isa nonconverter.

40. The method according to embodiment 38 or 39, wherein said screeningis carried out using a sequence selected from the group consisting ofSEQ ID NOS:1, 3, 35, 36, 37, and 38.

41. The method according to any one of embodiments 38-40, furthercomprising screening said DNA sample, or another DNA sample from saidtobacco plant of interest, for the presence of a mutation in SEQ IDNO:14, the presence of a mutation in SEQ ID NO:26, or the presence of amutation in SEQ ID NO:14 and SEQ ID NO:26.

42. An isolated polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of:

-   -   a) a nucleotide sequence comprising SEQ ID NO:1, 3, or 4;    -   b) a nucleotide sequence comprising a fragment of at least 20        consecutive nucleotides of SEQ ID NO:1, 3, or 4;    -   c) a nucleotide sequence having at least 97% sequence identity        to the entirety of the sequence set forth in SEQ ID NO:1,        wherein said polynucleotide encodes a polypeptide involved in        the metabolic conversion of nicotine to nornicotine in a plant;    -   d) a nucleotide sequence encoding a polypeptide selected from        the group consisting of SEQ ID NOS:2 and 5-13, or a fragment        thereof comprising at least 115 contiguous residues;    -   e) a nucleotide sequence encoding a polypeptide having at least        98% sequence identity to the sequence set forth in SEQ ID NO:2,        5, 6, 7, 8, 9, 10, 11, 12, or 13;    -   and    -   f) a nucleotide sequence that is complementary to the sequence        according to any of preceding items (a) through (e).

43. An isolated polypeptide comprising an amino acid sequence selectedfrom the group consisting of:

-   -   a) an amino acid sequence set forth in SEQ ID NO:2, 5, 6, 7, 8,        9, 10, 11, 12, or 13;    -   b) an amino acid sequence that is at least 98% identical to an        amino acid sequence set forth in SEQ ID NO:2, 5, 6, 7, 8, 9, 10,        11, 12, or 13; and    -   c) an amino acid sequence that is a fragment of the amino acid        sequence set forth in SEQ ID NO:2, 5, 6, 7, 8, 9, 10, 11, 12, or        13, wherein said fragment comprises at least 115 contiguous        residues of the amino acid sequence of SEQ ID NO:2, 5, 6, 7, 8,        9, 10, 11, 12, or 13.

44. A tobacco plant, or plant part thereof that is homozygous for amutation in a gene encoding a CYP82E10 nicotine demethylase, a geneencoding a CYP82E4 nicotine demethylase, and a gene encoding a CYP82E5nicotine demethylase, wherein said mutation results in reducedexpression or function of said CYP82E10, CYP82E4, and CYP82E5 nicotinedemethylase, wherein said CYP82E10 nicotine demethylase comprises amutation at position 381, said CYP82E4 nicotine demethylase comprises amutation at position 329, and said CYP82E5 nicotine demethylasecomprises a mutation at position 422, wherein said numbering isaccording to SEQ ID NO:2, 14, and 26, respectively.

45. A mutation in a gene encoding a CYP82E10 nicotine demethylase,wherein said mutation results in reduced expression or function of saidCYPe2E10 nicotine demethylase.

46. A plant having a mutation in a CYP82E10 gene that inhibits nicotinedemethylase activity in roots, a mutation in a CYP82E4v2 gene thatinhibits nicotine demethylase activity in senescent leaves, and amutation in a CYP83E5 gene that inhibits nicotine demethylase activityin green leaves.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-C shows the DNA (SEQ ID NO:4) and predicted protein sequences ofthe CYP82E10 nicotine demethylase gene. The protein coding sequences arein uppercase, and 5′ and 3′ flanking sequences are in lowercase. Theintron sequence (SEQ ID NO:3) is lowercase italicized. Numbers for thenucleotide sequence are shown on the left and numbers for the proteinsequence are labeled on the right. Nucleotide sequences corresponding tothe PCR primers used to specifically amplify exon 1 for mutationscreening are underlined (not shown in bold), whereas underlinedsequences in bold denote the exon 2-specific primer sites. Individualnucleotide and amino acid residues that were found to be altered in themutation screen (Table 2) are underlined and in bold.

FIG. 2A-C shows an alignment of genomic sequences for CYP82E10 (SEQ IDNO:4), CYP82E5v2 (SEQ ID NO:38), and CYP82E4v2 (SEQ ID NO:37).Protein-encoding sequences are in upper case type; 5′ and 3′untranslated regions are indicated in lower case type; and intronsequences are shown in lower case italicized type. Positions of sharedsequence identity are box shaded.

FIG. 3 shows thin layer chromatographic data of nicotine demethylaseactivities of microsomal membranes from yeast cells expressing CYP82E10,and CYP82E10 possessing the Pro381Ser (P381S) mutation from plant 1041.CPM, counts per minute.

FIGS. 4A and 4B show mean percent nicotine conversion for burley tobaccoplants with varying mutant combinations at CYP82E4v2, CYP82E5v2, andCYP82E10 loci. Means with different letters are significantly differentat the P<0.05 level.

DESCRIPTION OF THE SEQUENCES OF THE SEQUENCE LISTING

The following listing sets forth the sequence information for theSequence Listing. Standard notation for amino acid substitutions isused. Thus, for example, CYP82E10 P419S indicates the variant proteinhas a serine substitution for the proline residue at position 419, wherethe numbering is with respect to the wild-type sequence, in this case,the CYP82E10 sequence set forth in SEQ ID NO:2. As another example,CYP82E4 P38L indicates the variant protein has a leucine substitutionfor the proline residue at position 38, where the numbering is withrespect to the wild-type sequence, in this case, the CYP82E4 sequenceset forth in SEQ ID NO:14. As yet another example, CYP82E5 P72Lindicates the variant protein has a leucine substitution for the prolineresidue at position 72, where the numbering is with respect to thewild-type sequence, in this case, the CYP82E5 sequence set forth in SEQID NO:26.

SEQ ID NO:1 sets forth a coding sequence for CYP82E10.

SEQ ID NO:2 sets forth the amino acid sequence for CYP82E10.

SEQ ID NO:3 sets forth the nucleotide sequence of an intron of theCYP82E10 gene.

SEQ ID NO:4 sets forth the genomic sequence for CYP82E10.

SEQ ID NO:5 sets forth the amino acid sequence for CYP82E10 L148F.

SEQ ID NO:6 sets forth the amino acid sequence for CYP82E10 G172R.

SEQ ID NO:7 sets forth the amino acid sequence for CYP82E10 A344T.

SEQ ID NO:8 sets forth the amino acid sequence for CYP82E10 A410T.

SEQ ID NO:9 sets forth the amino acid sequence for CYP82E10 R417H.

SEQ ID NO:10 sets forth the amino acid sequence for CYP82E10 P419S.

SEQ ID NO:11 sets forth the amino acid sequence for CYP82E10 G79S.

SEQ ID NO:12 sets forth the amino acid sequence for CYP82E10 P107S.

SEQ ID NO:13 sets forth the amino acid sequence for CYP82E10 P381S.

SEQ ID NO:14 sets forth the amino acid sequence for CYP82E4.

SEQ ID NO:15 sets forth the amino acid sequence for CYP82E4 P38L.

SEQ ID NO:16 sets forth the amino acid sequence for CYP82E4 D171N.

SEQ ID NO:17 sets forth the amino acid sequence for CYP82E4 E201K.

SEQ ID NO:18 sets forth the amino acid sequence for CYP82E4 R169Q.

SEQ ID NO:19 sets forth the amino acid sequence for CYP82E4 G459R.

SEQ ID NO:20 sets forth the amino acid sequence for CYP82E4 T427I.

SEQ ID NO:21 sets forth the amino acid sequence for CYP82E4 V376M.

SEQ ID NO:22 sets forth the amino acid sequence for CYP82E4 W329Stop.

SEQ ID NO:23 sets forth the amino acid sequence for CYP82E4 K364N.

SEQ ID NO:24 sets forth the amino acid sequence for CYP82E4 P381S.

SEQ ID NO:25 sets forth the amino acid sequence for CYP82E4 P458S.

SEQ ID NO:26 sets forth the amino acid sequence for CYP82E5.

SEQ ID NO:27 sets forth the amino acid sequence for CYP82E5 P72L.

SEQ ID NO:28 sets forth the amino acid sequence for CYP82E5 L143F.

SEQ ID NO:29 sets forth the amino acid sequence for CYP82E5 S174L.

SEQ ID NO:30 sets forth the amino acid sequence for CYP82E5 M224I.

SEQ ID NO:31 sets forth the amino acid sequence for CYP82E5 P235S.

SEQ ID NO:32 sets forth the amino acid sequence for CYP82E5 A410V.

SEQ ID NO:33 sets forth the amino acid sequence for CYP82E5 W422Stop.

SEQ ID NO:34 sets forth the amino acid sequence for CYP82E5 P449L.

SEQ ID NO:35 sets forth the forward primer sequence for exon 1 ofCYP82E10.

SEQ ID NO:36 sets forth the reverse primer sequence for exon 1 ofCYP82E10.

SEQ ID NO:37 sets forth the forward primer sequence for exon 2 ofCYP82E10.

SEQ ID NO:37 sets forth the reverse primer sequence for exon 2 ofCYP82E10.

SEQ ID NO:38 sets forth the genomic sequence for CYP82E4v2.

SEQ ID NO:39 sets forth the genomic sequence for CYP82E5v2.

Definitions

The present invention includes compositions and methods for inhibitingexpression or function of root-specific nicotine demethylasepolypeptides that are involved in the metabolic conversion of nicotineto nornicotine in the roots of a plant, particularly plants of theNicotiana genus, including tobacco plants of various commercialvarieties.

As used herein, “inhibit,” “inhibition” and “inhibiting” are defined asany method known in the art or described herein, which decreases theexpression or function of a gene product of interest (i.e., the targetgene product), in this case a nicotine demethylase, such as aroot-specific nicotine demethylase of the invention. It is recognizedthat nicotine demethylase polypeptides can be inhibited by any suitablemethod known in the art, including sense and antisense suppression, RNAisuppression, knock out approaches such as mutagenesis, and the like. Ofparticular interest are methods that knock out, or knock down,expression and/or function of these root-specific nicotine demethylases,particularly mutagenic approaches that allow for selection of favorablemutations in the CYP82E10 nicotine demethylase gene.

By “favorable mutation” is intended a mutation that results in asubstitution, insertion, deletion, or truncation of the CYP82E10polypeptide such that its nicotine demethylase activity is inhibited. Insome embodiments, the nicotine demethylase activity is inhibited by atleast 25%, 30%, 35, 40%, 45, 50%, 55%, or 60% when compared to theactivity of the wild-type CYP82E10 polypeptide under the same testconditions. In other embodiments, the nicotine demethylase activity isinhibited by at least 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In preferredembodiments, the favorable mutation provides for complete inhibition(i.e., 100% inhibition), and the nicotine demethylase activity isknocked out (i.e., its activity cannot be measured).

“Inhibiting” can be in the context of a comparison between two plants,for example, a genetically altered plant versus a wild-type plant. Thecomparison can be between plants, for example, a wild-type plant and oneof which lacks a DNA sequence capable of producing a root-specificnicotine demethylase that converts nicotine to nornicotine. Inhibitionof expression or function of a target gene product also can be in thecontext of a comparison between plant cells, organelles, organs, tissuesor plant parts within the same plant or between different plants, andincludes comparisons between developmental or temporal stages within thesame plant or plant part or between plants or plant parts.

“Inhibiting” can include any relative decrement of function orproduction of a gene product of interest, in this case, a root-specificnicotine demethylase, up to and including complete elimination offunction or production of that gene product. When levels of a geneproduct are compared, such a comparison is preferably carried outbetween organisms with a similar genetic background. Preferably, asimilar genetic background is a background where the organisms beingcompared share 50% or greater, more preferably 75% or greater, and, evenmore preferably 90% or greater sequence identity of nuclear geneticmaterial. A similar genetic background is a background where theorganisms being compared are plants, and the plants are isogenic exceptfor any genetic material originally introduced using planttransformation techniques or a mutation generated by human intervention.Measurement of the level or amount of a gene product may be carried outby any suitable method, non-limiting examples of which include, but arenot limited to, comparison of mRNA transcript levels, protein or peptidelevels, and/or phenotype, especially the conversion of nicotine tonornicotine. As used herein, mRNA transcripts can include processed andnon-processed mRNA transcripts, and polypeptides or peptides can includepolypeptides or peptides with or without any post-translationalmodification.

As used herein, “variant” means a substantially similar sequence. Avariant can have different function or a substantially similar functionas a wild-type polypeptide of interest. For a nicotine demethylase, asubstantially similar function is at least 99%, 98%, 97%, 95%, 90%, 85%,80%, 75%, 60%, 50%, 25% or 15% of wild-type enzyme function ofconverting nicotine to nornicotine under the same conditions or in anear-isogenic line. A wild-type CYP82E10 is set forth in SEQ ID NO:2. Awild-type CYP82E4 is set forth in SEQ ID NO:14. A wild-type CYP82E5 isset forth in SEQ ID NO:26. Exemplary variants of the wild-type CYP82E10of the present invention include polypeptides comprising the sequenceset forth in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, or 13. The variant setforth in SEQ ID NO:10 (CYP82E10 P419S) advantageously has a favorablemutation that results in the enzyme having only about 25% of thenicotine demethylase activity of the wild-type CYP82E10 polypeptide. Thevariants set forth in SEQ ID NOs: 11 (CYP82E10 G79S), 12 (CYP82E10 withP107S), and 13 (CYP82E10 with P381S) advantageously have favorablemutations that result in their nicotine demethylase activity beingknocked out (i.e., 100% inhibition, and thus a nonfunctionalpolypeptide). In like manner, exemplary variants of the wild-typeCYP82E4 include polypeptides comprising the sequence set forth in SEQ IDNO:15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. The variant set forthin SEQ ID NO:21 (CYP82E4 V376M) advantageously has a favorable mutationthat results in the enzyme having only about 50% of the nicotinedemethylase activity of the wild-type CYP82E4 polypeptide. The variantsset forth in SEQ ID NOs: 22 (CYP82E4 W329Stop), 23 (CYP82E4 K364N), 24(CYP82E4 P381S), and 25 (CYP82E4 P458S) advantageously have favorablemutations that result in their nicotine demethylase activity beingknocked out (i.e., 100% inhibition). Similarly, exemplary variants ofthe wild-type CYP82E4 include polypeptides comprising the sequence setforth in SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, or 34. The variant setforth in SEQ ID NO:34 (CYP82E5 P449L) advantageously has a favorablemutation that results in inhibition of its nicotine demethylaseactivity, and the variant set forth in SEQ ID NO:33 advantageously has afavorable mutation that results in its nicotine demethylase activitybeing knocked out (i.e., 100% inhibition).

As used herein, a “variant polynucleotide” or “variant polypeptide”means a nucleic acid or amino acid sequence that is not wild-type.

A variant can have one addition, deletion or substitution; two or lessadditions, deletions or substitutions; three or less additions,deletions or substitutions; four or less additions, deletions orsubstitutions; or five or less additions, deletions or substitutions. Amutation includes additions, deletions, and substitutions. Suchdeletions or additions can be at the C-terminus, N-terminus or both theC- and N-termini Fusion polypeptides or epitope-tagged polypeptides arealso included in the present invention. “Silent” nucleotide mutations donot change the encoded amino acid at a given position Amino acidsubstitutions can be conservative. A conservative substitution is achange in the amino acid where the change is to an amino acid within thesame family of amino acids as the original amino acid. The family isdefined by the side chain of the individual amino acids. A family ofamino acids can have basic, acidic, uncharged polar or nonpolar sidechains. See, Alberts et al., (1994) Molecular biology of the cell (3rded., pages 56-57, Garland Publishing Inc., New York, N.Y.), incorporatedherein by reference as if set forth in its entirety. A deletion,substitution or addition can be to the amino acid of another CYP82Efamily member in that same position. As used herein, a “fragment” meansa portion of a polynucleotide or a portion of a polypeptide and henceprotein encoded thereby.

As used herein, “plant part” means plant cells, plant protoplasts, plantcell tissue cultures from which a whole plant can be regenerated, plantcalli, plant clumps and plant cells that are intact in plants or partsof plants such as embryos, pollen, anthers, ovules, seeds, leaves,flowers, stems, branches, fruit, roots, root tips and the like. Progeny,variants and mutants of regenerated plants are also included within thescope of the present invention, provided that they comprise theintroduced polynucleotides of the invention. As used herein, “tobaccoplant material” means any portion of a plant part or any combination ofplant parts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel nicotine demethylase gene,CYP82E10 (genomic sequence set forth in SEQ ID NO:4), and its encodedCYP82E10 nicotine demethylase (SEQ ID NO:2), that is involved inroot-specific conversion of nicotine to nornicotine in roots of tobaccoplants and its use in reducing or minimizing nicotine to nornicotineconversion and thus reducing levels of nornicotine in tobacco plants andplant parts thereof. By “root-specific” is intended it is preferentiallyexpressed within the roots of tobacco plants, as opposed to other plantorgans such as leaves or seeds. By introducing selected favorablemutations into this root-specific nicotine demethylase or variantsthereof having nicotine demethylase activity, in combination with one ormore selected favorable mutations within a gene encoding a green-leafnicotine demethylase (for example, CYP82E5 set forth in SEQ ID NO:26) orvariant thereof having nicotine demethylase activity, and further incombination with one or more selected favorable mutations within a geneencoding a senescence-induced nicotine demethylase (for example, CYP82E4set forth in SEQ ID NO:14) or variant thereof having nicotinedemethylase activity, it is possible to produce nontransgenic tobaccoplants having minimal nicotine to nornicotine conversion, where theconversion rate is less than about 1.5%, preferably less than about 1%.

Lowering nornicotine levels in tobacco is highly desirable because thisalkaloid serves as a precursor to the well-documented carcinogenN′-nitrosonornicotine (NNN). Two genes encoding proteins having nicotinedemethylase activity in tobacco have been previously identified anddesignated as CYP82E4v2 and CYP82E5v2. The CYP82E4 polypeptide (SEQ IDNO:14) is a senescence-induced nicotine demethylase. The CYP82E4v2 gene(including the coding and intron regions), its role in nornicotineproduction in tobacco plants, and methods for inhibiting its expressionand function are described in U.S. patent application Ser. No.11/580,765, which published as U.S. Patent Application Publication No.2008/0202541 A1. The CYP82E5 polypeptide (SEQ ID NO:26) is a green-leafnicotine demethylase (i.e., its predominant expression is in greenleaves). The CYP82E4 gene (including the coding and intron regions), itsrole in nornicotine production in tobacco plants, and methods forinhibiting its expression and function are described in U.S. patentapplication Ser. No. 12/269,531, which published as U.S. PatentApplication Publication No. 2009/0205072 A1. The contents of these twoU.S. patent applications and their respective publications are hereinincorporated by reference in their entirety.

Plants homozygous for favorable mutant cyp82e4v2 and cyp82e5v2 alleles(i.e., mutant alleles that knock down, or knock out, expression of theserespective nicotine demethylase genes), however, can still metabolizemore than 2% of their nicotine to nornicotine, which representnornicotine levels that can still lead to substantial NNN formation. Thediscovery of the CYP82E10 nicotine demethylase gene provides a furtheravenue for minimizing the nicotine to nornicotine conversion rate intobacco plants, and thus further reducing the levels of nornicotine andthus NNN in tobacco plants and plant materials derived therefrom.Combining favorable mutant cyp82e10 alleles with favorable mutantcyp82e4v2 and cyp82e5v2 alleles provides for tobacco plants possessingmore than a 3-fold reduction in nornicotine when compared to thatobserved for tobacco plants having the cyp82e4v2 mutation alone, or thecyp82e5v2 mutations together. In one embodiment, the present inventionprovides a homozygous triple mutant combination of nicotine demethylasegenes cyp82e4v2, cyp82e5v2, and cyp82e10) that results in nontransgenictobacco plants that produce very low levels of nornicotine comparable tothat only previously achieved via transgenic gene suppressionapproaches, such as those described in U.S. Patent ApplicationPublication Nos. 2008/0202541 A1 and 2009/0205072 A1.

Nicotine Demethylase Polynucleotides and Polypeptides, and Variants andFragments Thereof

Compositions of the present invention include the CYP82E10 polypeptideand variants and fragments thereof. Such nicotine demethylasepolynucleotides and polypeptides are involved in the metabolicconversion of nicotine to nornicotine in plants, including commercialvarieties of tobacco plants. In particular, compositions of theinvention include isolated polypeptides comprising the amino acidsequences as shown in SEQ ID NOs:2, and 5-13, isolated polynucleotidescomprising the nucleotide sequences as shown in SEQ ID NOs:1, 3, and 4,and isolated polynucleotides encoding the amino acid sequences of SEQ IDNOs:2 and 5-13. The polynucleotides of the present invention can finduse in inhibiting expression of nicotine demethylase polypeptides orvariants thereof that are involved in the metabolic conversion ofnicotine to nornicotine in plants, particularly tobacco plants. Some ofthe polynucleotides of the invention have mutations which result ininhibiting the nicotine demethylase activity of the wild-type nicotinedemethylase. The inhibition of polypeptides of the present invention iseffective in lowering nornicotine levels in tobacco lines where geneticconversion occurs in less than 30%, 50%, 70%, 90% of the population,such as flue-cured tobaccos. The inhibition of polypeptides of thepresent invention is effective in lowering nornicotine levels in tobaccopopulations where genetic conversion occurs in at least 90%, 80%, 70%,60%, 50% of a plant population. A population preferably contains greaterthan about 25, 50, 100, 500, 1,000, 5,000, or 25,000 plants where, morepreferably at least about 10%, 25%, 50%, 75%, 95% or 100% of the plantscomprise a polypeptide of the present invention.

The nicotine demethylase polynucleotides and encoded polypeptides of thepresent invention include a novel cytochrome P450 gene, designated theCYP82E10 nicotine demethylase gene, that is newly identified as having arole in the metabolic conversion of nicotine to nornicotine in roots oftobacco plants. Transgenic approaches such as sense, antisense, and RNAisuppression may be used to knock down expression of this nicotinedemethylase, in a manner similar to that described for the CYP82E4 andCYP82E5 nicotine demethylases, as described in U.S. Patent ApplicationPublication Nos. 2008/0202541 A1 and 2009/0205072 A1, the disclosures ofwhich are herein incorporated by reference in their entirety. Thepreferred approach is one that introduces one or more favorablemutations into this gene, as this approach advantageously providesnontransgenic tobacco plants having reduced nicotine to nornicotineconversion rates, and thus reduced levels of nornicotine and NNN. Suchapproaches include, but are not limited to, mutagenesis, and the like,as described elsewhere herein below.

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions of the present invention. An“isolated” or “purified” polynucleotide or protein, or biologicallyactive portion thereof, is substantially or essentially free fromcomponents that normally accompany or interact with the polynucleotideor protein as found in its naturally occurring environment. Thus, anisolated or purified polynucleotide or protein is substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived. A protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) ofcontaminating protein. When the protein of the invention or biologicallyactive portion thereof is recombinantly produced, optimally culturemedium represents less than about 30% 20%, 10%, 5%, or 1% (by dryweight) of chemical precursors or non-protein-of-interest chemicals.

Fragments of the disclosed polynucleotides and polypeptides encodedthereby are also encompassed by the present invention. Fragments of apolynucleotide may encode protein fragments that retain the biologicalactivity of the native protein and hence are involved in the metabolicconversion of nicotine to nornicotine in a plant. Alternatively,fragments of a polynucleotide that are useful as hybridization probes orPCR primers generally do not encode fragment proteins retainingbiological activity. Furthermore, fragments of the disclosed nucleotidesequences include those that can be assembled within recombinantconstructs for use in gene silencing with any method known in the art,including, but not limited to, sense suppression/cosuppression,antisense suppression, double-stranded RNA (dsRNA) interference, hairpinRNA interference and intron-containing hairpin RNA interference,amplicon-mediated interference, ribozymes, and small interfering RNA ormicro RNA, as described in the art and herein below. Thus, fragments ofa nucleotide sequence may range from at least about 20 nucleotides,about 50 nucleotides, about 70 nucleotides, about 100 nucleotides about150 nucleotides, about 200 nucleotides, 250 nucleotides, 300nucleotides, and up to the full-length polynucleotide encoding theproteins of the invention, depending upon the desired outcome. In oneaspect, the fragments of a nucleotide sequence can be a fragment between100 and about 350 nucleotides, between 100 and about 325 nucleotides,between 100 and about 300 nucleotides, between about 125 and about 300nucleotides, between about 125 and about 275 nucleotides in length,between about 200 to about 320 contiguous nucleotides, between about 200and about 420 contiguous nucleotides in length between about 250 andabout 450 contiguous nucleotides in length. Another embodiment includesa recombinant nucleic acid molecule having between about 300 and about450 contiguous nucleotides in length.

A fragment of a nicotine demethylase polynucleotide of the presentinvention that encodes a biologically active portion of a CYP82E10polypeptide of the present invention will encode at least 15, 25, 30,50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500contiguous amino acids, or up to the total number of amino acids presentin a full-length nicotine demethylase polypeptide of the invention(e.g., 517 amino acids for SEQ ID NOs:2 and 5-13). A biologically activeportion of a nicotine demethylase polypeptide can be prepared byisolating a portion of one of the CYP82E10 polynucleotides of thepresent invention, expressing the encoded portion of the CYP82E10polypeptide (e.g., by recombinant expression in vitro), and assessingthe activity of the encoded portion of the CYP82E10 polypeptide, i.e.,the ability to promote conversion of nicotine to nornicotine, usingassays known in the art and those provided herein below.

Polynucleotides that are fragments of a CYP82E10 nucleotide sequence ofthe present invention comprise at least 16, 20, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 950, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, or 1700 contiguous nucleotides, or up to the number of nucleotidespresent in a full-length CYP82E10 polynucleotide as disclosed herein(e.g., 1551 for SEQ ID NO: 1; 2636 for SEQ ID NO:4). Polynucleotidesthat are fragments of a CYP82E10 nucleotide sequence of the presentinvention comprise fragments from about 20 to about 1700 contiguousnucleotides, from about 50 to about 1600 contiguous nucleotides, fromabout 75 to about 1500 contiguous nucleotides, from about 100 to about1400 nucleotides, from about 150 to about 1300 contiguous nucleotides,from about 150 to about 1200 contiguous nucleotides, from about 175 toabout 1100 contiguous nucleotides, about 200 to about 1000 contiguousnucleotides, about 225 to about 900 contiguous nucleotides, about 500 toabout 1600 contiguous nucleotides, about 775 to about 1700 contiguousnucleotides, about 1000 to about 1700 contiguous nucleotides, or fromabout 300 to about 800 contiguous nucleotides from a CYP82E10polynucleotide as disclosed herein. In one aspect, fragmentpolynucleotides comprise a polynucleotide sequence containing thepolynucleotide sequence from the nucleotide at about position 700 toabout position 1250 of a CYP82E10 coding sequence, at about position 700to about position 1250 of a CYP82E10 genomic sequence, at about position10 to about position 900 of a CYP82E10 intron sequence, or at aboutposition 100 to about position 800 of a CYP82E10 intron sequence.

Variants of the disclosed polynucleotides and polypeptides encodedthereby are also encompassed by the present invention. Naturallyoccurring variants include those variants that share substantialsequence identity to the CYP82E10 polynucleotides and polypeptidesdisclosed herein as defined herein below. In another embodiment,naturally occurring variants also share substantial functional identityto the CYP82E10 polynucleotides disclosed herein. The compositions andmethods of the invention can be used to target expression or function ofany naturally occurring CYP82E10 that shares substantial sequenceidentity to the disclosed CYP82E10 polypeptides. Such CYP82E10polypeptides can possess the relevant nicotine demethylase activity,i.e., involvement in the metabolic conversion of nicotine to nornicotinein plants, or not. Such variants may result from, for example, geneticpolymorphism or from human manipulation as occurs with breeding andselection, including mutagenesis approaches. Biologically activevariants of a CYP82E10 protein of the invention, for example, variantsof the polypeptide set forth in SEQ ID NO:2 and 5-13, will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the aminoacid sequence for the wild-type protein as determined by sequencealignment programs and parameters described elsewhere herein, and can becharacterized by their functional involvement in the metabolicconversion of nicotine to nornicotine in plants, or lack thereof. Abiologically active variant of a protein of the invention may differfrom that protein by as few as 1-15 amino acid residues, as few as 10,as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, as fewas 4, as few as 3, as few as 2, or as few as 1 amino acid residue. Abiologically inactive variant of a protein of the invention may differfrom that protein by as few as 1-15 amino acid residues, as few as 10,as few as 9, as few as 8, as few as 7, as few as 6, as few as 5, as fewas 4, as few as 3, as few as 2, or as few as 1 amino acid residue.

Variants of a particular polynucleotide of the present invention includethose naturally occurring polynucleotides that encode a CYP82E10polypeptide that is involved in the metabolic conversion of nicotine tonornicotine in the roots of plants. Such polynucleotide variants cancomprise a deletion and/or addition of one or more nucleotides at one ormore sites within the native polynucleotide disclosed herein and/or asubstitution of one or more nucleotides at one or more sites in thenative polynucleotide. Because of the degeneracy of the genetic code,conservative variants for polynucleotides include those sequences thatencode the amino acid sequence of one of the CYP82E10 polypeptides ofthe invention. Naturally occurring variants such as these can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as are known in the art and disclosed herein. Variantpolynucleotides also include synthetically derived polynucleotides, suchas those generated, for example, by using site-directed mutagenesis butwhich still share substantial sequence identity to the naturallyoccurring sequences disclosed herein, and thus can be used in themethods of the invention to inhibit the expression or function of anicotine demethylase that is involved in the metabolic conversion ofnicotine to nornicotine, including the nicotine demethylase polypeptidesset forth in SEQ ID NOS:2, 5, 6, 7, 8, 9, and 10. Generally, variants ofa particular polynucleotide of the invention, for example, thepolynucleotide sequence of SEQ ID NO:3 or the polynucleotide sequenceencoding the amino acid sequence set forth in SEQ ID NO:2, and 5-13,will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to that particular polynucleotide as determined by sequencealignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide of the present invention (alsoreferred to as the reference polynucleotide) can also be evaluated bycomparison of the percent sequence identity between the polypeptideencoded by the reference polynucleotide and the polypeptide encoded by avariant polynucleotide. Percent sequence identity between any twopolypeptides can be calculated using sequence alignment programs andparameters described elsewhere herein. Where any given pair ofpolynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

Furthermore, the polynucleotides of the invention can be used to isolatecorresponding root-specific nicotine demethylase sequences, particularlyCYP82E10 sequences, from other members of the Nicotiana genus. PCR,hybridization, and other like methods can be used to identify suchsequences based on their sequence homology to the sequences set forthherein. Sequences isolated based on their sequence identity to thenucleotide sequences set forth herein or to variants and fragmentsthereof are encompassed by the present invention. Such sequences includesequences that are orthologs of the disclosed sequences.

According to the present invention, “orthologs” are genes derived from acommon ancestral gene and which are found in different species as aresult of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions oforthologs are often highly conserved among species. Thus, isolatedpolynucleotides that encode for a nicotine demethylase polypeptide thatis involved in the nicotine-to-nornicotine metabolic conversion andwhich hybridize under stringent conditions to the CYP82E10 sequencedisclosed herein, or to variants or fragments thereof, are encompassedby the present invention. Such sequences can be used in the methods ofthe present invention to inhibit expression of nicotine demethylasepolypeptides that are involved in the metabolic conversion of nicotineto nornicotine in plants.

Using PCR, oligonucleotide primers can be designed for use in PCRreactions to amplify corresponding DNA sequences from cDNA or genomicDNA extracted from any plant of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed,Cold Spring Harbor Laboratory Press, Plainview, N.Y.). Innis et al.,eds. (1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like.

Hybridization techniques involve the use of all or part of a knownpolynucleotide as a probe that selectively hybridizes to othercorresponding polynucleotides present in a population of cloned genomicDNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism.

Hybridization may be carried out under stringent conditions. By“stringent conditions” or “stringent hybridization conditions” isintended conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. Stringent conditions may also beachieved with the addition of destabilizing agents such as formamideExemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

In a specific embodiment, stringency conditions include hybridization ina solution containing 5×SSC, 0.5% SDS, 5×Denhardt's, 0.45 ug/ul Poly ARNA, 0.45 ug/ul calf thymus DNA and 50% formamide at 42° C., and atleast one post-hybridization wash in a solution comprising from about0.01×SSC to about 1×SSC. The duration of hybridization is from about 14to about 16 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However; severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m) those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m), of less than 45° C. (aqueous solution)or 32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

Hybridization probes may be genomic DNA fragments, cDNA fragments, RNAfragments, or other oligonucleotides, and may be labeled with adetectable group such as ³²P, or any other delectable marker. Forexample, probes for hybridization can be made by labeling syntheticoligonucleotides based on the CYP82E10 polynucleotides sequences of thepresent invention. Methods for preparation of probes for hybridizationand for construction of cDNA and genomic libraries are generally knownin the art and are disclosed in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

For example, the CYP82E10 polynucleotide sequences disclosed herein, orone or more portions thereof, may be used as probes capable ofspecifically hybridizing to corresponding root-specific nicotinedemethylase polynucleotides and messenger RNAs. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique among the CYP82E10 polynucleotide sequences orunique to one of the CYP82E10 polynucleotide sequences, includingupstream regions 5′ to the coding sequence and downstream regions 3′ tothe coding sequence and an intron region (for example, SEQ ID NO:3), andare optimally at least about 10 contiguous nucleotides in length, moreoptimally at least about 20 contiguous nucleotides in length, moreoptimally at least about 50 contiguous nucleotides in length, moreoptimally at least about 75 contiguous nucleotides in length, and moreoptimally at least about 100 contiguous nucleotides in length. Suchprobes may be used to amplify corresponding CYP82E10 polynucleotides.This technique may be used to isolate additional coding sequences ormutations from a desired plant or as a diagnostic assay to determine thepresence of coding sequences in a plant. Hybridization techniquesinclude hybridization screening of plated DNA libraries (either plaquesor colonies; see, for example, Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

As used herein, with respect to the sequence relationships between twoor more polynucleotides or polypeptides, the term “reference sequence”is a defined sequence used as a basis for sequence comparison. Areference sequence may be a subset or the entirety of a specifiedsequence; for example, as a segment of a full-length cDNA or genesequence, or the complete cDNA or gene sequence.

As used herein, the term “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, where thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the deference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 arebased on the algorithm of Karlin and Altschul (1990) supra. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleotidesequence encoding a protein of the invention. BLAST protein searches canbe performed with the BLASTX program, score=50. wordlength=3, to obtainamino acid sequences homologous to a protein or polypeptide of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST2.0) can be used to perform an iterated search that detects distantrelationships between molecules. See Altschul et al. (1997) supra. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTX forproteins) can be used (See www.ncbi.nlna.nih.gov). Alignment may also beperformed manually by inspection.

In some embodiments, the sequence identity/similarity values providedherein are calculated using the BLASTX (Altschul et al. (1997) supra),Clustal W (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680), andGAP (University of Wisconsin Genetic Computing Group software package)algorithms using default parameters. The present invention alsoencompasses the use of any equivalent program thereof for the analysisand comparison of nucleic acid and protein sequences. By “equivalentprogram” is intended any sequence comparison program that, for any twosequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby BLASTX. Clustal W, or GAP.

For purposes of the foregoing discussion of variant nucleotide andpolypeptide sequences encompassed by the present invention, the term“sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for malting this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

The term “percentage of sequence identity” as used herein means thevalue determined by comparing two optimally aligned sequences over acomparison window, where the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Thus, CYP82E10 polynucleotide and polypeptide sequences can beidentified using the sequences provided herein. Such methods includeobtaining a polynucleotide or polypeptide sequence at least 80%, 85%,90%, 95%, 98%, 99% sequence identity with the polynucleotide sequence ofSEQ ID NO: 1, 3, or 4 or a complement or fragment thereof, or apolypeptide sequence of SEQ ID NO: 2, or 5-13. A preferred embodimentincludes a polypeptide corresponding to SEQ ID NO:2 that has a serine atposition 79, 107, or 381 of the CYP82E10 polypeptide, where thenumbering corresponds to SEQ ID NO:2.

Methods for Inhibiting Expression or Function of a Nicotine Demethylase

Methods of reducing the concentration, content, and/or activity of aCYP82E10 polypeptide of the present invention in a Nicotiana plant orplant part, particularly the root tissue, are provided. Many methods maybe used, alone or in combination, to reduce or eliminate the activity ofthe CYP82E10 polypeptide of the present invention (SEQ ID NO:2), andvariants thereof that retain nicotine demethylases activity (forexample, SEQ ID NOs:7, 8, 9, and 10). In addition, combinations ofmethods may be employed to reduce or eliminate the activity of two ormore different nicotine demethylases, more particularly theroot-specific CYP82E10 nicotine demethylase and one or both of thegreen-leaf CYP82E5 and senescence-induced CYP82E4 nicotine demethylases.In a particular embodiment, the CYP82E5 is a polypeptide with at leastone amino acid mutation in the sequence of SEQ ID NO: 26 that negativelyaffects conversion in green leaves and the CYP82E4 has the sequence setforth in SEQ ID NO:14 with at least one amino acid mutation thatnegatively affects conversion in senescent leaves.

In accordance with the present invention, the expression of a CYP82E10nicotine demethylase of the present invention is inhibited if theprotein level of the CYP82E10 polypeptide is statistically lower thanthe protein level of the same CYP82E10 polypeptide in a plant that hasnot been genetically modified or mutagenized to inhibit the expressionof that CYP82E10 polypeptide, and where these plants have been culturedand harvested using the same protocols. In particular embodiments of theinvention, the protein level of the CYP82E10 polypeptide in a modifiedplant according to the invention is less than 95%, less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, or less than 5% of theprotein level of the same CYP82E10 polypeptide in a plant that is not amutant or that has not been genetically modified to inhibit theexpression of that CYP82E10 polypeptide and which has been cultured andharvested using the same protocols. The expression level of the CYP82E10polypeptide may be measured directly, for example, by assaying for thelevel of the CYP82E10 transcript or CYP82E10 polypeptide expressed inthe tobacco plant or plant part, or indirectly, for example, bymeasuring the conversion of nicotine to nornicotine in the tobacco plantor plant part. Methods for monitoring the expression level of a proteinare known in the art, and include, but are not limited to, Northern blotanalysis and RNA differentiation assays. Methods for determining theactivity of a targeted CYP82E10 polypeptide in converting nicotine tonornicotine are known in the art and described elsewhere herein below,and include, but are not limited to, alkaloid analysis using gaschromatography.

The present invention provides methods for reducing the level ofnornicotine, or reducing the rate of conversion of nicotine tonornicotine, in a tobacco plant, or plant part thereof. The methodscomprise introducing into the genome of a tobacco plant a mutationwithin at least one allele of each of at least three nicotinedemethylase genes, wherein the mutation reduces expression of thenicotine demethylase gene, and wherein a first of these nicotinedemethylase genes encodes a root-specific nicotine demethylase involvedin the metabolic conversion of nicotine to nornicotine in a tobaccoplant or a plant part thereof. In some embodiments, the root-specificnicotine demethylase is CYP82E10 or variant thereof. In otherembodiments, these methods comprise introducing into the genome of atobacco plant a mutation within at least one allele of a nicotinedemethylase gene encoding CYP82E10 or variant thereof, and a mutationwithin at least one allele of a nicotine demethylase encoding CYP82E4 orvariant thereof, and/or a nicotine demetyylase encoding CYP82E5 orvariant thereof.

A number of approaches have been used to combine mutations in one plantincluding sexual crossing. A plant having a favorable mutation in aCYP82E10 gene that inhibits the nicotine demethylases activity in rootscan be crossed with a plant having a favorable mutation in a CYP82E4v2gene that inhibits the nicotine demethylase activity in senescentleaves, or be crossed with a plant having a favorable mutation in aCYP83E5v2 gene that inhibits nicotine demethylase activity in greenleaves to produce a plant having reduced nicotine to nornicotineconversion. In preferred embodiments, crosses are made in order tointroduce a favorable mutation within a CYP82E10, CYP82E4v2, andCYP82E5v2 gene within the same plant. In this manner, a plant having afavorable mutation in a CYP82E10 gene that inhibits the nicotinedemethylases activity in roots is crossed with a plant having afavorable mutation in a CYP82E4v2 gene that inhibits the nicotinedemethylase activity in senescent leaves and a favorable mutation in aCYP83E5v2 gene that inhibits nicotine demethylase activity in greenleaves. Alternatively, a plant having a favorable mutation in aCYP82E4v2 gene that inhibits the nicotine demethylase activity insenescent leaves is crossed with a plant having a favorable mutation ina CYP82E10 gene that inhibits the nicotine demethylase activity in rootsand a favorable mutation in a CYP83E5v2 gene that inhibits nicotinedemethylase activity in green leaves. In yet another embodiment, a planthaving a favorable mutation in a CYP82E5v2 gene that inhibits thenicotine demethylase activity in green leaves is crossed with a planthaving a favorable mutation in a CYP82E10 gene that inhibits thenicotine demethylase activity in roots and a favorable mutation in aCYP83E4v2 gene that inhibits nicotine demethylase activity in senescentleaves. By introducing a favorable mutation into each of these nicotinedemethylases genes it is possible to produce a plant having reducednicotine to nornicotine conversion rates with conversion levels lowerthan about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%.

In a more preferred embodiment, a plant having one or more favorablemutations that results in a modification of the CYP82E10 polypeptide atposition 79, 107, 381, or 419 (where the numbering is according to SEQID NO:2) can be crossed with a plant having one or more favorablemutations that results in a modification of the CYP82E4 polypeptide atposition 329, 364, 376, 381, or 458 and/or having one or more favorablemutations that results in a modification of the CYP82E5 polypeptide atposition 422 or 449 to produce a plant with conversion levels lower than0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%. A particularly preferredconversion level of nicotine to nornicotine can be between 0.05%-0.4%,between 0.1-0.6%, between 0.1%-0.3%, between 0.1%-0.5%, between0.1%-0.4%, between 0.1%-0.7%, between 0.1%-1.0%, between 0.1%-1.1%,between 0.1%-1.2%, between 0.1%-1.3%, between 0.1%-1.4%, or between0.1%-1.5%. Any mutation of a polynucleotide of the present inventionthat results in a truncation of the CYP82E10, CYP83E4, or CYP83E5polypeptide before a conserved heme-binding motif will inhibit theenzyme and can be used in a cross described above. The domains ofcytochrome P450 proteins are known in the art. See, for example, Xu etal. (2007) Physiologia Plantarum 129:307-319, hereby incorporated byreference. By crossing plants having a nonfunctional or inhibitedCYP82E10 gene with plants having a nonfunctional or inhibited CYP82E4v2gene, a nonfunctional or inhibited CYP82E5v2 gene, or nonfunctional orinhibited CYP82E4v2 and CYP82E5v2 genes, nornicotine levels can bereduced in a tobacco plant.

The activity of a CYP82E10, CYP82E4, or CYP82E5 nicotine demethylasepolypeptide in converting nicotine to nornicotine in a tobacco plant orplant part is inhibited according to the present invention if thisconversion activity is statistically lower than conversion activity ofthe same nicotine demethylase polypeptide in a tobacco plant or plantpart that has not been genetically modified to inhibit the conversionactivity of that nicotine demethylase polypeptide and which has beencultured and harvested using the same protocols. In particularembodiments, activity of a nicotine demethylase polypeptide inconverting nicotine to nornicotine in a modified tobacco plant or plantpart according to the invention is inhibited if the activity is lessthan 95%, less than 90%, less than 80% less than 70%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20% less than10%, less than 5%, less than 2%, or less than 1% of the conversionactivity of the same nicotine demethylase polypeptide in a tobacco plantthat has not been genetically modified to inhibit the expression of thatnicotine demethylase polypeptide and has been cultured and harvestedusing the same protocols. The activity of a nicotine demethylasepolypeptide in converting nicotine to nornicotine in a tobacco plant orplant part is eliminated according to the invention when it is notdetectable by the assay methods described elsewhere herein. Methods ofdetermining the activity of a nicotine demethylase polypeptide inconverting nicotine to nornicotine in a tobacco plant using gaschromatography are disclosed in the examples here in below.

In some embodiments, the favorable mutation is introduced into a tobaccoplant or plant part using a mutagenesis approach, and the introducedmutation is selected using methods known to those of skill in the artsuch as, but not limited to, Southern blot analysis, DNA sequencing, PCRanalysis, or phenotypic analysis. A plant or plant part altered ormodified by the foregoing embodiments is grown under plant formingconditions for a time sufficient to modulate the concentration and/oractivity of polypeptides of the present invention in the plant. Plantforming conditions are well known in the art and discussed brieflyelsewhere herein.

A modified tobacco plant containing a favorable mutation in a nicotinedemethylase described herein has a reduced level of conversion ofnicotine to nornicotine. In particular embodiments, conversion ofnicotine to nornicotine in a modified tobacco plant or plant partaccording to the invention is less than 95%, less than 90%, less than80% less than 70%, less than 60%, less than 50%, less than 40%, lessthan 30%, less than 20% less than 10%, less than 5%, less than 2%, orless than 1% of the conversion in a tobacco plant that that has not beengenetically modified to inhibit the expression of that nicotinedemethylase polypeptide and which has been cultured and harvested usingthe same protocols. In some embodiments, the modified tobacco plant is aconverter tobacco plant. In other embodiments, the modified tobaccoplant is a nonconverter tobacco plant. In some embodiments, the modifiedtobacco plant has a conversion rate lower than the rate observed incommercial non-converter tobacco plants.

According to the present invention, changes in levels, ratios, activity,or distribution of CYP82E10 polypeptides of the present invention, orchanges in tobacco plant or plant part phenotype, particularly reducedaccumulation of nornicotine and its carcinogenic metabolite, NNN, couldbe measured by comparing a subject plant or plant part to a controlplant or plant part, where the subject plant or plant part and thecontrol plant or plant part have been cultured and/or harvested usingthe same protocols. As used herein, a subject plant or plant part is onein which genetic alteration, for example, by mutagenesis, has beenaffected as to the nicotine demethylase polypeptide of interest, or is atobacco plant or plant part that is descended from a tobacco plant orplant part so altered and which comprises the alteration. A controlplant or plant part provides a reference point for measuring changes inphenotype of the subject plant or plant part. The measurement of changesin phenotype can be measured at any time in a plant or plant part,including during plant development, senescence, or after curing. Inother embodiments, the measurement of changes in phenotype can bemeasured in plants grown under any conditions, including from plantsgrown in growth chamber, greenhouse, or in a field. In one embodiment,changes in phenotype can be measured by determining the nicotine tonornicotine conversion rate. In a preferred embodiment, conversion canbe measured by dividing the percentage of nornicotine (as a percentageof the total tissue weight) by the sum of the percentage nicotine andnornicotine (as percentages of the total tissue weight) and multiplyingby 100.

According to the present invention, a control plant or plant part maycomprise a wild-type tobacco plant or plant part, i.e., of the samegenotype as the starting material for the genetic alteration thatresulted in the subject plant or plant part. A control plant or plantpart may also comprise a tobacco plant or plant part of the samegenotype as the starting material but that has been transformed with anull construct (i.e., with a construct that has no known effect on thetrait of interest, such as a construct comprising a selectable markergene). In all such cases, the subject plant or plant part and thecontrol plant or plant part are cultured and harvested using the sameprotocols.

In some embodiments, the activity of a nicotine demethylase polypeptideof the present invention may be reduced or eliminated by disrupting thegene encoding the nicotine demethylase polypeptide. The inventionencompasses mutagenized plants that carry mutations in nicotinedemethylase genes, where the mutations reduce expression of the nicotinedemethylase gene or inhibit the activity of an encoded nicotinedemethylase polypeptide of the present invention.

In other embodiments, the activity of a nicotine demethylase polypeptideof the present invention is reduced or eliminated by disrupting the geneencoding the nicotine demethylase polypeptide. The gene encoding thenicotine demethylase polypeptide may be disrupted by any method known inthe art, for example, by transposon tagging or by mutagenizing plantsusing random or targeted mutagenesis and selecting for plants that havereduced nicotine demethylase activity or mutations in CYP82E10, alone orin combination with mutations in CYP82E4 or CYP82E5.

Transposon tagging may be used to reduce or eliminate the activity ofone or more CYP82E10 nicotine demethylase polypeptides of the presentinvention. Transposon tagging comprises inserting a transposon within anendogenous nicotine demethylase gene to reduce or eliminate expressionof the nicotine demethylase polypeptide. Methods for the transposontagging of specific genes in plants are well known in the art. See, forexample, Maes et al. (1999) Trends Plant Sci. 4:90-96; Dharmapuri andSonti (1999) FEMS Micerobiol. Lett. 179:53-59; Meissner et al. (2000)Plant J. 22:265-274; Phogat et al. (2000) J. Biosci. 25:57-63; Walbot(2000) Curr. Opin. Plant Biol. 2:103-107; Gai et al. (2000) NucleicAcids Res. 28:94-9b; Fitzmaurice et al. (1999) Genetics 153:1919-1928).

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, using mutagenic or carcinogenic compoundsincluding ethyl methanesulfonate-induced mutagenesis, deletionmutagenesis, and fast neutron deletion mutagenesis used in a reversegenetics sense (with PCR) to identify plant lines in which theendogenous gene has been deleted. For examples of these methods seeOhshima et al. (1998) Virology 213:472-481; Okubara et al. (1994)Genetics 137:867-874; and Quesada et al. (2000) Genetics 154:421-4315;each of which is herein incorporated by reference. In addition, a fastand automatable method for screening for chemically induced mutations,TILLING (Targeting Induced Local Lesions In Genomes), using denaturingHPLC or selective endonuclease digestion of selected PCR products isalso applicable to the instant invention. See McCallum et al (2000) Nat.Biotechnol. 18:455-457, herein incorporated by reference.

Mutations that impact gene expression or that interfere with thefunction of the encoded nicotine demethylase protein can be determinedusing methods that are well known in the art. Insertional mutations ingene exons usually result in null-mutants. Mutations in conservedresidues can be particularly effective in inhibiting the metabolicfunction of the encoded protein. Conserved residues of plant nicotinedemethylase polypeptides suitable for mutagenesis with the goal toeliminate activity of a nicotine demethylase polypeptide in convertingnicotine to nornicotine in a tobacco plant or plant part have beendescribed. See FIG. 1A-C of U.S. Patent Application Publication No.2009/0205072 A1, herein incorporated by reference in its entirety, wherethe residues that differ from the other P450 polypeptides are shaded ingrey. The conserved residue is that which is not shaded in grey at eachposition. Such mutants can be isolated according to well-knownprocedures.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba et al. (2003) Plant Cell15:1455-1467.

In another embodiment of the invention, the compositions of theinvention find use in screening methods to identify nonconverter plantsfor use in breeding programs. In this manner, the nucleotide sequencesof the invention can be used to screen native germplasms fornonconverter plants having a stable mutation in the CYP82E10 geneidentified herein. These nonconverter plants identified by the methodsof the invention can be used to develop breeding lines.

In addition to the nucleotide sequences encoding the CYP82E10polypeptides described herein, compositions of the invention include anintron sequence in the CYP82E10 gene sequence that can be used inscreening methods. While not bound by any mechanism of action, theCYP82E10 gene(s) may represent the only member(s) of the cytochrome P450family involved in the metabolic conversion of nicotine to nornicotinein roots of tobacco. For certain applications it would be useful to havea means of diagnostically differentiating this specific member of thecytochrome P450 gene family from the rest of the closely relatedsequences within this family. For example, it is possible that withinthe naturally existing tobacco germplasm (or in mutagenizedpopulations), accessions may exist in which this gene is naturallydysfunctional and may therefore may be valuable as a permanentlynonconverter resource. Such dysfunctional sequences may include thoseencoding the polypeptides set forth in SEQ ID NO: 11, 12, or 13. Amethod to specifically assay for such genotypes (e.g. deletion mutants,rearrangements, and the like) could serve as a powerful tool. Thepresent invention includes primers designed to specifically amplify exon1 and exon 2 of CYP82E10 where one of the two primer pairs correspondsto the intron between the exons. Examples of primers useful to amplifythe exons of CYP82E10 include SEQ ID NO: 35 with SEQ ID NO: 36 and SEQID NO: 37 with SEQ ID NO: 38. These same primers can be used forsequence analysis of the products.

Because the intron regions of genes are typically less conserved thanexons, it is predicted that the use of an intron-specific probe wouldbetter enable one to distinguish the gene(s) corresponding to theCYP82E10 gene from the other members of the CYP82E family. The use of aCYP82E10 intron-specific probe, and/or the PCR primers used to generateproducts provide powerful tools in assays to determine whether anynaturally occurring, or mutagenized, tobacco plants possess deletions orrearrangements that may render the gene inactive. Such a plant can thenbe used in breeding programs to create tobacco lines that are incapableof converting.

Tobacco Plants, Plant Parts, and Products Having Reduced Nornicotine andNNN Content

The CYP82E10 polynucleotides of the invention, and variants andfragments thereof, can be used in the methods of the present inventionto inhibit expression or function of CYP82E10 nicotine demethylases thatare involved in the metabolic conversion of nicotine to nornicotine in aplant. In this manner, favorable mutations can be introduced into theCYP82E10 gene of interest. The methods of the invention do not depend ona particular method for introducing the favorable mutation into theCYP82E10 nicotine demethylase gene.

The compositions and methods of the invention can be used to reduce thenornicotine content, particularly in the leaves and stems, of any plantof the genus Nicotiana including, but not limited to, the followingspecies: acuminata, affinis, alata, attenuate, bigelovii, clevelandii,excelsior, forgetiana, glauca, glutinosa, langsdorffii, longiflora,obtusifolia, palmeri, paniculata, plumbaginifolia, qudrivalvis, repanda,rustica, suaveolens, sylvestris, tabacum, tomentosa, trigonophylla, andx sanderae. The present invention can also be practiced using anyvarieties of a plant of the genus Nicotiana, including but not limitedto Nicotiana acuminata multiflora, Nicotiana alata grandiflora,Nicotiana bigelovii quadrivalvis, Nicotiana bigelovii wallacei,Nicotiana obtusifolia obtusifolia, Nicotiana obtusifolia plameri,Nicotiana quadrivalvis bigelovii, Nicotiana quadrivalvis quadrivalvis,Nicotiana quadrivalvis wallacei, and Nicotiana trigonophylla palmeri, aswell as varieties commonly known as flue or bright varieties, Burleyvarieties, dark varieties and oriental/Turkish varieties. In someembodiments, the tobacco plant of interest is a Burley, Va., flue-cured,air-cured, fire-cured, Oriental, or a dark tobacco plant.

The tobacco plants and varieties described herein are suitable forconventional growing and harvesting techniques, such as cultivation inmanure rich soil or without manure, bagging the flowers or no bagging,or topping or no topping. The harvested leaves and stems may be used inany traditional tobacco product including, but not limited to, pipe,cigar and cigarette tobacco, and chewing tobacco in any form includingleaf tobacco, shredded tobacco, or cut tobacco.

Thus the present invention provides a tobacco plant, or plant partthereof, comprising a mutation in a gene encoding a CYP82E10 nicotinedemethylase, wherein said mutation results in reduced expression orfunction of said CYP82E10 nicotine demethylases, and a reduced amount ofnornicotine and N′-nitrosonornicotine. As used herein, the term “areduced amount” or “a reduced level” is intended to refer to an amountof nornicotine and/or N′-nitrosonornicotine in a plant of the presentinvention or a plant part or tobacco product thereof that is less thanwhat would be found in a plant of the genus Nicotiana or a plant part ortobacco product from the same variety of tobacco, processed (i.e.,cultured and harvested) in the same manner, that has not beengenetically modified for reduced nornicotine and/orN′-nitrosonornicotine. The amount of nornicotine may be reduced by about10% to greater than about 90%, including greater than about 20%, about30%, about 40%, about 50%, about 60%, about 70%, and about 80%. Theconversion of nicotine to nornicotine can be less than 0.3%, less than0.5%, less than 0.7%, between 0.1%-0.5%, between 0.1%-0.4%, between0.1%-0.7%, or between 0.1%-1.0% in plants, plant parts, and products ofthe present invention, and more specifically in plants, plant partshaving mutations in CYP82E10, CYP82E4v2, and CYP825v2.

The term “tobacco products” as used herein include, but are not limitedto, smoking materials (e.g., any cigarette, including a cigarillo, anon-ventilated or vented recess filter cigarette, a cigar, pipetobacco), smokeless products (e.g., snuff, chewing tobacco,biodegradable inserts (e.g., gum, lozenges, dissolving strips)). See,for example, U.S. Patent 2005/0019448, herein incorporated by reference.The present invention also encompasses a range of tobacco product blendsthat can be made by combining conventional tobacco with differingamounts of the low nornicotine and/or N′-nitrosonornicotine tobaccodescribed herein. In further embodiments, the plant or plant part of thegenus Nicotiana as described above is cured tobacco.

In some embodiments of the present invention, the tobacco productreduces the carcinogenic potential of tobacco smoke that is inhaleddirectly with consumption of a tobacco product such as cigars,cigarettes, or pipe tobacco, or inhaled as secondary smoke (i.e., by anindividual that inhales the tobacco smoke generated by an individualconsuming a tobacco product such as cigars, cigarettes, or pipetobacco). The cured tobacco described herein can be used to prepare atobacco product, particularly one that undergoes chemical changes due toheat, comprising a reduced amount of nornicotine and/orN′-nitrosonornicotine in the smoke stream that is inhaled directly orinhaled as secondary smoke. In the same manner, the tobacco products ofthe invention may be useful in the preparation of smokeless tobaccoproducts such as chewing tobacco, snuff and the like.

The tobacco products derived from the tobacco plants of the presentinvention thus find use in methods for reducing the carcinogenicpotential of these tobacco products, and reducing the exposure of humansto the carcinogenic nitrosamine NNN, particularly for individuals thatare users of these tobacco products. The following examples are offeredby way of illustration and not by way of limitation.

EXPERIMENTAL

The citations mentioned in the following discussion are provided at theclose of the Experimental section.

Background

The knowledge that CYP82E4v2 represents the nicotine demethylase locusresponsible for the high nornicotine accumulation observed in Converterplants (Siminszky et al., 2005), opened the door for nontransgenic, aswell as transgenic, approaches toward overcoming the conversion problemand lowering the nornicotine content of the senescent, cured leaf.Specifically, it became possible for researchers to generate tobaccopopulations that had been exposed to a chemical mutagen, and select forindividuals possessing nonfunctional alleles at the CYP82E4v2 locus. Infact, three independent groups have already generated nonconvertingtobacco lines based on this strategy (Dewey et al., 2007; Xu et al.,2007b; Julio et al., 2008).

As previously reported, a tobacco plant designated 775 was identifiedfrom an EMS-mutagenized population of Burley line DH98-325-6 and shownto possess a knockout mutation within the CYP82E4v2 gene (Dewey et al.,2007). In the summer of 2008, plants homozygous for the 775 mutationwere grown at the Upper Coastal Plains research station in Rocky Mount,N.C., and air-cured according to standard industry practice. Alkaloidanalysis of these materials was conducted using the “LC Protocol”described by Jack et al. (2007). As shown in Table 1, plants possessingthe 775 mutation averaged 2.6% nicotine to nornicotine conversion, Incontrast, >60% conversion was observed in the parental line DH98-325-6,a strong converter genotype. Nearly identical results were reported byJulio et al. (2008), who recorded conversion percentages ranging from2.82 to 3.37 for plants homozygous for a cyp82e4v2 knockout mutantwithin the strong converter burley genotype BB16NN (parental conversionrates ranged between 68-98%). Thus, debilitating mutations in CYP82E4v2alone appear to be effective in eliminating the problems arising fromthe unstable genetic phenomenon associated with the generation ofConverter plants.

TABLE 1 Alkaloid profiles for experimental materials evaluated in 2008field experiment. Percentage values represent an average. Gene AminoAcid % % % % % Genotype Targeted Mutation^(b) Change Nicotine^(c)Nornicotine Anabasine Anatabine Conversion^(d) DH98-325-6 control(15)^(a) Control — — 1.228 2.014 0.016 0.125 62.4 TN90LC (14) Control —— 4.680 0.157 0.022 0.155 3.2 DH98-325-6 RNAi CYP82E4v2 — — 3.351 0.0400.016 0.101 1.2 300-08 #1 (15) and related DH98-325-6 RNAi CYP82E4v2 — —3.741 0.026 0.017 0.106 0.7 300-02 #1 (15) and related DH98-325-6 #775CYP82E4v2 G986A W329Stop 2.941 0.077 0.013 0.093 2.6 Homo. (15)DH98-325-6 #1013 CYP82E5v2 G1266A W422Stop 1.005 1.876 0.012 0.097 65.2Homo. (14) DH98-325-6 Double Double Double 3.160 0.076 0.015 0.117 2.3Homozygous Mutant (9) ^(a)Number in parentheses indicates total numberof plants analyzed. ^(b)Numbering relative to start codon of cDNAsequence. ^(c)Percentages were calculated on a dry tobacco weight basis.^(d)Percentage nicotine conversion equals [% nornicotine/(%nornicotine + % nicotine)] × 100.

Although the utilization of tobacco plants possessing the 775, orcomparable, mutations in CYP82E4v2 can be an effective means ofeliminating the introduction of Converter plants within tobaccopopulations, a low, but significant amount of nornicotine remains inthese plants. Given that nicotine to nornicotine conversion rates as lowas 0.45% were observed in transgenic plants expressing an RNAi-basedconstruct directed against CYP82E4v2 (Lewis et al., 2008), it wasapparent that at least one other gene with high DNA sequence homology toCYP82E4v2 must be responsible for the majority of the nornicotinesynthesis that is observed within both Nonconverter plants and Converterplants possessing an inactivated CYP82E4v2 gene. This possibility wasfurther supported by the discovery of CYP82E5v2, a gene that shares92.7% DNA sequence identity with CYP82E4v2 that was also shown to encodea functional nicotine demethylase enzyme (Dewey et al., 2007; Gavilanoand Siminszky, 2007). The CYP82E5v2 nicotine demethylase gene isexpressed at low levels in green tobacco leaves of Converter andNonconverter plants alike, in contrast to CYP82E4v2 which is expressedat very high levels, but only in the leaves of Converter plants duringsenescence and air-curing.

As outlined in Dewey et al. (2007), screening of an EMS-mutagenizedDH98-325-6 tobacco population lead to the identification of anindividual (plant 1013) possessing a knockout mutation in CYP82E5v2. Todetermine the impact of the non-functional cyp82e5v2 allele onnornicotine accumulation, crosses were made that combined the mutationsfrom plants 775 and 1013. Molecular genotyping of numerous F₂individuals derived from the F₁ progeny of the initial cross resulted inthe identification of nine individuals that were homozygous for bothmutations (e4e4/e5e5). These nine plants were also included in the 2008field trial. Despite the fact the CYP82E5v2 has been shown to encode afunctional nicotine demethylase enzyme (Dewey et al., 2007; Gavilano andSiminszky, 2007), combining the dysfunctional cyp82e5v2 mutation withthe knockout cyp82e4v2 mutation had remarkably little impact on leafnornicotine levels. As shown in Table 1, plants homozygous for thedouble mutation (e4e4/e5e5) averaged 2.3% nicotine conversion, comparedwith an average of 2.6% conversion for plants possessing only thecyp82e4v2 mutation (e4e4). The modest difference in mean conversionbetween the two genotypes was not statistically significant (P=0.118).In contrast, one of the CYP82E4v2 RNAi-silenced transgenic lines thatwas included in this study averaged 0.7% conversion, an amountsignificantly lower (P<0.001) than that obtained from either the e4e4 ore4e4/e5e5 genotypes. Thus, another gene with high homology to CYP82E4v2must exist within the tobacco genome that contributes toward nornicotineproduction in the plant.

Example 1: Isolation and Characterization of the cyp82e10 NicotineDemethylase Gene

To identify other genes in the tobacco genome that have the potential ofencoding nicotine demethylase enzymes, homology searches using theBLASTN and BLASTX algorithms (Altschul et al., 1990, 1997) were directedagainst the N. tabacum expressed sequenced tagged (EST) databases inGenBank, using the DNA and protein sequences of CYP82E4v2 as therespective query sequences. In addition to identifying cDNA sequencescorresponding to previously characterized members of the CYP82Esuperfamily (such as CYP82E2, CYP82E3 and CYP82E5v2), seven ESTs werediscovered that did not align perfectly with any previouslycharacterized member of this gene family Interestingly, all seven of theESTs originated from either root-specific cDNA libraries, or cDNAlibraries made up of mixed tissues that included roots. This observationsuggested that the new CYP82E gene is expressed specifically in roottissue, a property that could explain why this particular member of theCYP82E P450 superfamily has eluded detection previously, as priorefforts have focused on the characterization of CYP82E genes expressedin leaf tissue. Because no individual EST sequence was long enough tocover the entire coding region of this novel gene, PCR primers weredesigned that enabled amplification of the entire cDNA sequence fromfirst-strand cDNA that had been generated from RNA isolated from tobaccoroot tissue. In addition, primers were used to amplify the correspondinggenomic region of the gene that includes a central, large intron. Thisnovel CYP82E cDNA shares 92.4% nucleotide identity with the tobaccoCYP82E4v2 cDNA, and a 91.1% predicted identity at the amino acid level.In keeping with the guidelines for P450 gene nomenclature, this new genewas designated CYP82E10. Of all the characterized members of the CYP82Esuperfamily, CYP82E10 displays that highest sequence similarity withCYP82E5v2, sharing 96.5% nucleotide identity at the cDNA level and 95.7%predicted amino acid sequence identity. The DNA sequence of CYP82E10 andits predicted protein sequence are shown in FIG. 1.

Although the cDNAs of the various CYP82E family members tend to behighly conserved, the genomic versions of these genes show much greatersequence diversity. This is due primarily to the substantial sequencedivergence observed within the large, central intron. An alignment ofCYP82E4v2, CYP82E5v2, and CYP82E10 genomic sequences is shown in FIG. 2.As calculated using the EMBOSS Pairwise Alignment algorithm(www.ebi.ac.uk/Tools/emboss/align/index.html), the CYP82E4v2 andCYP82E10 genes share 78.3% nucleotide identity, and CYP82E10 is 84.9%identical to the CYP82E5v2 gene as they exist within the tobacco genome(CYP82E4v2 and CYP82E5v2 genomic sequences share 75% identity).

As detailed in several publications, most of the genes of the CYP82Esuperfamily that are found in the tobacco genome do not encodefunctional nicotine demethylase enzymes (Siminszky et al., 2005;Chakrabarti et al., 2007; Dewey et al., 2007; Gavilano et al., 2007; Xuet al., 2007a). Therefore, sequence homology alone is not a veryaccurate indicator of gene function for the CYP82E family Instead,expression analysis in either transgenic plants (Siminszky et al., 2005)or in yeast (Gavilano and Siminszky, 2007; Xu et al., 2007a) has becomethe established means for determining whether individual members of thisgene family encode nicotine demethylase activity.

To determine whether CYP82E10 functions as a nicotine demethylase gene,its cDNA was cloned into the yeast expression vector pYeDP60 andtransformed into yeast strain W(R). Strain W(R) is a yeast cell linethat was engineered to overexpress the yeast NADPH-dependent P450reductase, an enzyme that serves as the direct electron donor to P450s;this system greatly enhances the detection of foreign P450 enzymeactivities that are expressed in yeast (Pompon et al., 1995). Nicotinedemethylase assays were conducted by incubating yeast microsomalmembrane preparations with [¹⁴C]-nicotine, and resolving the products bythin layer chromatography as described in Siminszky et al. (2005).

As shown in FIG. 3, no nicotine demethylase activity could be detectedusing yeast microsomes from the W(R) strain expressing only the pYeDP60vector. In contrast, a very robust nicotine demethylase activity couldbe measures from microsomes derived from yeast cells expressing theCYP82E10 cDNA. By measuring CYP82E10 enzyme activity across a wide rangeof [¹⁴C]-nicotine concentrations, a substrate saturation curve wasestablished and an apparent K_(m) of 3.9 μM nicotine was calculatedusing the microsomal assay. This kinetic parameter for CYP82E10 is verysimilar to the K_(m)s reported for the CYP82E4v2 and CYP82E5v2 enzymeswhen similarly expressed in yeast (Gavilano et al., 2007; Gavilano andSiminszky, 2007; Xu et al., 2007a).

Example 2: Identification of Plants Possessing Mutant Alleles ofCYP82E10

In order to accurately assess the specific contribution of CYP82E10toward the total nornicotine content of the tobacco plant, it wasnecessary to: (1) identify a tobacco plant with a knockout mutationwithin this gene; and (2) combine this mutation with the cyp82e4v2 andcyp82e5v2 mutations originating from plants 775 and 1013, respectively.To identify potentially debilitating mutations in CYP82E10, theEMS-mutagenized DH98-325-6 population was screened by high-throughputDNA sequence analysis using primers that specifically amplify portionsof CYP82E10 (without simultaneously amplifying other members of theCYP82E superfamily) To specifically amplify exon 1 of CYP82E10, thefollowing PCR primers were used: 5′-GTGATAGTTTGATTCCCAAGTGC-3′ (forward)and 5′-CTCCCAAAGTTAGATTAGTCCG-3′ (reverse); specific amplification ofexon 2 was achieved using the primers 5′-AGGTCGCGCTGATTCTTG-3′ (forward)and 5′-AGATGAATACCCATCTATCTAGGAGT-3′ (reverse). To ensure maximalspecificity, the reverse primer for exon 1 and the forward primer forexon 2 correspond to sequences within the CYP82E10 intron (FIG. 1). PCRamplification and sequence analysis of the mutagenized plants wasconducted using a 96-well format as described in Dewey et al. (2007).

High-throughput sequence analysis of over 1,200 individuals from themutagenized tobacco population resulted in the identification of 15individuals with mutations in CYP82E10. The most notable of these areshown in Table 2. The nucleotide and amino acid residues mutated inthese plants are also highlighted in FIG. 1. Although no truncationmutations were observed among these individuals, in several cases,mutations were identified that altered an amino acid residue within ahighly conserved region of the enzyme. To determine the effects of aparticular mutation on CYP82E10 enzyme activity, site-directedmutagenesis was used to introduce the specific mutations correspondingto seven of the nine mutations shown in Table 2 into the CYP82E10 cDNAwithin the pYeDP60 yeast expression vector. Microsomal preparations fromyeast strains expressing each of the seven CYP82E10 variants wereassayed in vitro for nicotine demethylase activity using bothnon-saturating (2.45 μM) and saturating (50 μM) concentrations of[¹⁴C]-nicotine. The results from the yeast expression assays showed thatmutations found plants 693, 817 and 1035 did not alter enzyme activity,whereas the mutations found in plants 1041, 1512 and 2476 lead tocomplete enzyme inactivation. The mutation observed in plant 1442resulted in a 75% reduction in activity compared to the wild typeCYP82E10 enzyme.

The thin layer chromatographic data for the in vitro yeast expressionassays corresponding to the plant 1041 mutation are shown in FIG. 3.This particular mutation was selected for more extensive investigation.To provide additional confirmation that the Pro to Ser substitution atamino acid position 381 that defines the plant 1041 mutation isincompatible with nicotine demethylation function, this same mutationwas introduced into a CYP82E4v2 cDNA that had been similarly cloned intothe pYeDP60 vector. The results of these yeast assays are displayed inTable 3. Whether introduced into the CYP82E10 or CYP82E4v2 enzymes, aSer substitution for Pro at position 381 leads to the complete ablationof nicotine demethylase activity in this assay. Interestingly, althoughthe activities of the wild type CYP82E10 and CYP82E4v2 enzymes werecomparable at the non-saturating [¹⁴C]-nicotine concentration (2.45 μM),at the 25 μM substrate level, the rate of [¹⁴C]-nornicotine synthesiswas nearly three times greater in microsomal preparations possessing theCYP82E10 enzyme than preparations containing CYP82E4v2.

TABLE 2 EMS treated lines of DH98-325-6 with mutations in the CYP82E10gene. Activity of Mutant Plant Number Mutation^(a) Amino Acid ChangeEnzyme^(b) 2476 G235A G79S Not detected 1512 C319T P107S Not detected319 C442T L148F Not tested 634 G514A G172R Not tested 1035 G1030A A344T100% 1041 C1141T P381S Not detected 817 G1228A A410T 100% 693 G1250AR417H 100% 1442 C1255T P419S  25% ^(a)In reference to the start codon ofthe CYP82E10 cDNA sequence. ^(b)Relative to the wild type enzyme whenexpressed in yeast.

TABLE 3 Nicotine demthylase activity of CYP82E4v2 and CYP82E10 enzymespossessing the 1041 mutation (Pro381Ser). CPM nornicotine CPMnornicotine at 2.45 μM at 50.0 μM [¹⁴C]-nicotine [¹⁴C]-nicotine Vectorsubstrate^(a) substrate pYeDP60-CYPE4v2 1,813 ± 623^(b)  5,383 ± 505pYeDP60-CYPE4v2/1041 Not detected Not detected pYeDP60-CYPE10 2,296 ±99   15,253 ± 465 pYeDP60-CYPE10/1041 Not detected Not detected^(a)Counts per minute of [¹⁴C]-nornicotine/mg microsomal protein.^(b)Standard deviation of two technical replications.

Nicotine demethylase activities of wild type and 1041 mutantCYP82E10-expressing yeast cells was also assayed in vivo. Yeast cultureswere shaken overnight in the presence of 55 μM [¹⁴C] nicotine, extractedwith methanol and analyzed by thin layer chromatography.[¹⁴C]-nornicotine could be detected in the extracts of yeast expressingwild type CYP82E10, but not the 1041 mutant version of the gene (datanot shown). Cumulatively, the yeast expression assays strongly suggestthat CYP82E10 enzyme function is completely abolished by theintroduction of the 1041 mutation.

Example 3: Combining Mutant Alleles of cyp82e10, cyp82e4v2 and cyp82e5v2

Given that the original 1041 mutation is in a genetic background(DH98-325-6) that contains both a strong converter CYP82E4v2 allele aswell as a wild type CYP82E5v2 gene, the only way to accurately assessthe specific contribution of CYP82E10 toward total plant nornicotinecontent is to introduce the 1041 mutation into tobacco plants possessingknockout CYP82E4v2 and CYP82E5v2 mutations as well. To accomplish this,plants heterozygous for the 1041 mutation (e10E10) were crossed withplants heterozygous for both the 775 and 1013 mutations described above(e4E4/e5E5). The latter plants represent progeny of from the cross775/1013//TN90/3/TN90/4/TN90. F₁ plants heterozygous for all threenicotine demethylase mutations (e4E4/e5E5/e10E10) were identified bymolecular genotyping, and allowed to self-pollinate. Moleculargenotyping was also used to screen over 400 F₂ progeny and subsequentlygroup them into the following genotypic classes: E4E4/E5E5/e10e10 (3plants total); e4e4/E5E5/e10e10 (4 plants total); E4E4/e5e5/e10e10 (5plants total); and e4e4/e5e5/e10e10 (5 plants total).

All of the plants described above were transplanted and grown in thefield at the Upper Coastal Plains research station in Rocky Mount, N.C.in the summer of 2009. Also included in this study were two of thegenotypes tested in the 2008 field trial shown in Table 1. Specifically,ten DH98-325-6 plants homozygous for only the cyp82e4v2 mutation(e4e4/E5E5/E10E10) and eleven DH98-325-6 plants possessing the doublehomozygous e4e4/e5e5/E10E10 genotype were included for comparison. Ascontrols, individual plants randomly selected from a commercial “lowconverter” seedlot (TN90LC), wild type DH98-325-6 individuals, andplants from one of the best CYP82E4v2 RNAi-suppressed transgenic lineswas also included in the study. After the plants were about an averageof 30 cm tall (35 days after transplanting) leaves from similar stalkpositions where collected, treated with ethephon and air-cured accordingto the protocol established by Jack et al. (2007). Alkaloid content ofthe cured leaf materials was determined by gas chromatography asdescribed in the same protocol.

Table 4 and FIG. 4 shows the results of the alkaloid analyses for the2009 field trial. Consistent with previous observations, the cyp82e4v2knockout mutation alone negates the strong converter phenotype of lineDH98-325-6, and also confers a substantially lower nornicotineaccumulation phenotype than plants from the commercial TN90LC seed (2.2%conversion versus 7.1%, respectively). As observed in the 2008 fieldtrial (Table 1), combining the cyp82e5v2 mutation with cyp82e4v2 did notlead to further reductions in nornicotine content. In fact, the meannicotine conversion for the e4e4/E5E5/E10E10 plants was actually lowerthan that observed for e4e4/e5e5/E10E10 individuals (2.2% versus 2.3%),though this slight difference was not statistically significant. Asexpected, the cyp82e10 mutation had no impact on the high nornicotinelevels conferred by an active CYP82E4v2 gene, either alone(E4E4/E5E5/e10e10 genotypes), or when combined with a mutant cyp82e5v2allele (E4E4/e5e5/e10e10 genotypes) (FIG. 4A) Similar to the cyp82e4v2and cyp82e5v2 double mutant results (Tables 1 and 4), introducingcyp82e10 into a cyp82e4v2 background was not effective in reducingnornicotine levels below than that which could be achieved by thecyp82e4v2 mutation alone (FIG. 4B). The e4e4/E5E5/e10e10 genotypesaveraged 1.85% conversion which was not significantly different than the2.2% mean conversion levels observed for e4e4/E5E5/E10E10 individuals(P=0.235).

TABLE 4 Alkaloid profiles for experimental materials evaluated in 2009field experiment. Measurements taken from leaves harvested 35 days aftertransplanting. Percentage values represent an average. Gene Amino Acid %% % % % Genotype Targeted Mutation^(b) Change Nicotine^(c) NornicotineAnabasine Anatabine Conversion^(d) DH98-325-6 control (8)^(a) Control —— 0.133 1.553 0.009 0.085 92.21 TN90LC (11) Control — — 1.519 0.1040.002 0.065 7.15 DH98-325-6 RNAi CYP82E4v2 — — 1.747 0.009 0.003 0.0630.54 300-02 #1 (10) and related DH98-325-6 #775 CYP82E4v2 G986A W329Stop1.375 0.030 0.002 0.057 2.20 Homo. (10) DH98-325-6 Double CYP82E4v2Double Double 1.524 0.036 0.003 0.084 2.34 Homo. Mutant (11) CYP82E5v2DH980325-6 #1041 CYP82E10 C1141T P381S 0.082 1.302 0.007 0.073 93.87Homo. (3) DH98-325-6 Double CYP82E5v2 Double Double 0.081 1.345 0.0100.068 94.31 Homo. Mutant (5) CYP82E10 DH98-325-6 Double CYP82E4v2 DoubleDouble 2.168 0.045 0.004 0.087 1.85 Homo. Mutant (4) CYP82E10 DH98-325-6Triple CYP82E4v2 Triple Triple 1.793 0.012 0.003 0.056 0.55 Homo. Mutant(5) CYP82E5v2 CYP82E10 ^(a)Number in parentheses indicates total numberof plants analyzed. ^(b)Numbering relative to start codon of cDNAsequence. ^(c)Percentages were calculated on a dry tobacco weight basis.^(d)Percentage nicotine conversion equals [% nornicotine/(%nornicotine + % nicotine)] × 100.

Although the cyp82e5v2 and cyp82e10 mutations did not serve tosignificantly decrease the nornicotine content of cyp82e4v2 plants whencombined individually, pyramiding all three nicotine demethylasemutations had a very notable effect. Nicotine to nornicotine conversionin triple mutant plants (e4e4/e5e5/e10e10) averaged only 0.55%, apercentage virtually identical to the 0.54% observed in theRNAi-suppressed transgenic line (P=0.893; FIG. 4B). This represents overa 3-fold reduction in nicotine conversion beyond that which was mediatedby the cyp82e4v2 mutation alone. Statistically, the differences inpercent nicotine conversion (and nornicotine accumulation as apercentage of total dry weight) between e4e4/E5E5/E10E10 ande4e4/e5e5/e10e10 genotypes was highly significant (P<0.0001). Similar tothe investigation of RNAi-mediated suppression of nicotine conversion(Lewis et al., 2008), the present nontransgenic alteration of nicotinedemethylase activities in the tobacco plant did not appear tosignificantly alter the content of the minor alkaloid species anatabineand anabasine.

The effects of pyramiding the three independent nicotine demethylasegene mutations were also tested in a field trial conducted during the2010 growing season. For this study, the crosses were conducted entirelywithin the DH98-325-6 genetic background (in contrast to the 2009 studywhere a TN90 parent was also used). Molecular genotyping was again usedto create every possible combination needed to determine the respectivecontributions of each CYP82E locus on the nornicotine phenotype.Alkaloid data were collected on tobacco plants that were grown tomaturity and cured according to standard industry practice. As shown inTable 5, a high level of nicotine conversion (ranging from 52.4-65.59%)was observed in all genotypes homozygous for a wild type CYP82E4v2 gene(genotypes E4E4/E5E5/E10E10, E4E4/e5e5/E10E10, E4E4/E5E51e10e10, andE4E4/e5e5/e10e10). Plants homozygous for just the cyp82e4v2 mutation(e4e4/E5E5/E10E10) averaged 2.91% nicotine to nornicotine conversion.Similar to the 2009 results, the effects of the cyp82E5v2 and cyp82E10mutations were not additive, and were only manifest when all threemutant loci were pyramided together. DH98-325-6 (e4e4/E5E5/e10e10)plants averaged 2.89% conversion and DH98-325-6 (e4e4/e5e5/E10E10)individuals averaged 2.52%, values that were not statistically differentthan that observed with the cyp82e4v2 mutation alone. In contrast, thereduction in nornicotine observed in the triple mutant DH98-325-6(e4e4/e5e5/e10e10) genotype (1.11% nicotine conversion) was 2.6-foldlower than that attained via the cyp82e4v2 mutation alone. The reductionin nicotine conversion attributable to the triple mutant combination washighly significant (P<0.001) compared with either cyp82e4v2 alone or anydouble mutant combination.

TABLE 5 Alkaloid profiles for DH98-325-6 genotypes possessing differentmutation combinations at the CYP82E4v2 (E4), CYP82Ev25 (E5), andCYP82E10 (E10) loci. Data are averaged over five replications andgenerated from analysis of composite ground samples of the fourth andfifth leaves from the top of the plant. Nicotine Nico- Conver- tineNornicotine Anabasine Anatabine sion Genotype (%) (%) (%) (%) (%)DH98-325-6 E4E4 1.76 2.46 0.02 0.17 58.66 E5E5 E10E10 DH98-325-6 e4e42.61 0.08 0.01 0.09 2.91 E5E5 E10E10 DH98-325-6 E4E4 1.08 2.06 0.02 0.1465.59 e5e5 E10E10 DH98-325-6 E4E4 1.40 1.96 0.01 0.13 59.30 E5E5 e10e10DH98-325-6 e4e4 3.25 0.09 0.02 0.16 2.89 e5e5 E10E10 DH98-325-6 e4e43.59 0.09 0.01 0.12 2.52 E5E5 e10e10 DH98-325-6 E4E4 1.59 1.72 0.01 0.0952.40 e5e5 e10e10 DH98-325-6 e4e4 4.18 0.05 0.02 0.13 1.11 e5e5 e10e10Alkaloid percentages were calculated on a dry weight basis Percentagenicotine conversion equals [% nornicotine/(% nornicotine + % nicotine)]× 100

CONCLUSIONS

Through the present discovery and characterization of a new nicotinedemethylase gene, CYP82E10, it has been possible to develop a strategyfor reducing the nicotine conversion rates (and thus nornicotine levels)in commercial grade air-cured tobacco plants to levels that havepreviously only been possible using transgenic approaches. This non-GMObased technology can reduce the levels of nornicotine to a degreesimilar to that which has been achieved using transgenic strategies, yetoffers the tremendous advantage of serving as a means for developingultra-low nornicotine tobacco varieties while bypassing the substantialhurdles associated with the commercialization of transgenic crops, suchas: (1) negotiating and paying licensing fees for the several enablingtechnologies required for generating transgenic plants; (2) avoiding thelengthy time and onerous costs associated with the deregulation of atransgenic event; and (3) encountering the possibility of productrejection by end users philosophically opposed to GMOs. The discoveryreported here represents a major advancement in our ability to lower thelevels of one of the most well documented strong carcinogens found intobacco products, in comparison with the previously described non-GMOstrategies that only targeted mutations in the CYP82E4v2 nicotinedemethylase gene (Julio et al., 2008; Xu et al., 2007b) or combinedCYP82E4v2 and CYP82E5v2 mutations (Dewey et al., 2007). Using transgenictechnologies, it was previously demonstrated that lowering nicotineconversion levels from ˜2.6% to ˜0.5% in the cured leaf lead to acommensurate reduction in the NNN content of the leaf as well (Lewis etal., 2008). One would expect to see similar reductions in the NNNcontent from tobacco leaves containing the triple mutant combination(e4e4/e5e5/e10e10) described in this report. Although originallytargeted for air-cured tobaccos, this technology will be of benefit toflue-cured varieties as well. As heat exchangers age, their ability toremove NO_(x) gases during flue-curing can decrease. Furthermore, recentstudies have shown that a considerable amount of TSNA formation canoccur during the storage of the cured leaf. Minimizing nornicotinelevels through the introduction of the triple mutant combination influe-cured varieties can act as a safeguard against NNN formation eitherduring storage or as a consequence of inefficient heat exchange duringthe curing process.

REFERENCES

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Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the list of embodiments andappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

1-46. (canceled)
 47. A Nicotiana tabacum plant comprising: (a) a firstmutation positioned between nucleotide 1872 and nucleotide 2483 ascompared to SEQ ID NO: 4 at a first locus encoding a CYP82E10 nicotinedemethylase having at least 95% sequence identity to the full length ofSEQ ID NO: 4; (b) a second mutation at a second locus encoding a CYP82E4nicotine demethylase, wherein the second mutation results in reducedexpression or function of the CY82E4 nicotine demethylase as compared toa control Nicotiana tabacum plant lacking the second mutation; and (c) athird mutation at a third locus encoding a CYP82E5v2 nicotinedemethylase, wherein the second mutation results in reduced expressionor function of the CY82E5vs nicotine demethylase as compared to acontrol Nicotiana tabacum plant lacking the third mutation.
 48. TheNicotiana tabacum plant of claim 47, wherein the first locus is 100%identical to SEQ ID NO:
 4. 49. The Nicotiana tabacum plant of claim 47,wherein the second mutation comprises a mutation at amino acid position329 according to SEQ ID NO: 14; and wherein the third mutation comprisesa mutation at amino acid position 422 according to SEQ ID NO:
 26. 50.The Nicotiana tabacum plant of claim 47, wherein the second mutation atthe second locus comprises a Tryptophan to stop codon substitution atamino acid position 329 of a CYP82E4 nicotine demethylase, wherein theCYP82E4 amino acid position numbering is according to SEQ ID NO: 14; andwherein the third mutation at the third locus comprises a Tryptophan tostop codon substitution at amino acid position 422 of a CYP82E5v2nicotine demethylase, wherein the CYP82E5v2 amino acid positionnumbering is according to SEQ ID NO:
 26. 51. The Nicotiana tabacum plantof claim 47, wherein the first mutation results in reduced expression orfunction of the CY82E10 nicotine demethylase as compared to a controlNicotiana tabacum plant lacking the first mutation.
 52. The Nicotianatabacum plant of claim 47, wherein the first mutation is selected fromthe group consisting of a point mutation, a deletion, an insertion, andan inversion.
 53. The Nicotiana tabacum plant of claim 47, wherein theNicotiana tabacum plant is of a tobacco type selected from the groupconsisting of a Burley type, a Virginia type, a bright type, a darktype, a flue-cured type, an air-cured type, a fire-cured type, a Turkishtype, and an Oriental type.
 54. A tobacco product comprising cured plantmaterial from the Nicotiana tabacum plant of claim
 47. 55. The tobaccoproduct of claim 54, wherein the tobacco product is selected from thegroup consisting of a cigar, a cigarette, pipe tobacco, a cigarillo, anon-ventilated or vented recess filter cigarette, a dissolving strip, agum, a tablet, snuff, and chewing tobacco.
 56. A Nicotiana tabacum seedfrom the Nicotiana tabacum plant of claim 47, wherein the seed comprisesthe first mutation, the second mutation, and the third mutation.
 57. ANicotiana tabacum plant comprising (a) a first mutation positionedbetween nucleotide 115 and nucleotide 1053 as compared to SEQ ID NO: 4at a first locus encoding a CYP82E10 nicotine demethylase having atleast 95% sequence identity to the full length of SEQ ID NO: 4; (b) asecond mutation at a second locus encoding a CYP82E4 nicotinedemethylase, wherein the second mutation results in reduced expressionor function of the CY82E4 nicotine demethylase as compared to a controlNicotiana tabacum plant lacking the second mutation; and (c) a thirdmutation at a third locus encoding a CYP82E5v2 nicotine demethylase,wherein the second mutation results in reduced expression or function ofthe CY82E5vs nicotine demethylase as compared to a control Nicotianatabacum plant lacking the third mutation.
 58. The Nicotiana tabacumplant of claim 57, wherein the first locus is 100% identical to SEQ IDNO:
 4. 59. The Nicotiana tabacum plant of claim 57, wherein the secondmutation comprises a mutation at amino acid position 329 according toSEQ ID NO: 14; and wherein the third mutation comprises a mutation atamino acid position 422 according to SEQ ID NO:
 26. 60. The Nicotianatabacum plant of claim 47, wherein the second mutation at the secondlocus comprises a Tryptophan to stop codon substitution at amino acidposition 329 of a CYP82E4 nicotine demethylase, wherein the CYP82E4amino acid position numbering is according to SEQ ID NO: 14; and whereinthe third mutation at the third locus comprises a Tryptophan to stopcodon substitution at amino acid position 422 of a CYP82E5v2 nicotinedemethylase, wherein the CYP82E5v2 amino acid position numbering isaccording to SEQ ID NO:
 26. 61. The Nicotiana tabacum plant of claim 57,wherein the first mutation results in reduced expression or function ofthe CY82E10 nicotine demethylase as compared to a control Nicotianatabacum plant lacking the first mutation.
 62. The Nicotiana tabacumplant of claim 57, wherein the first mutation is selected from the groupconsisting of a point mutation, a deletion, an insertion, and aninversion.
 63. The Nicotiana tabacum plant of claim 57, wherein theNicotiana tabacum plant is of a tobacco type selected from the groupconsisting of a Burley type, a Virginia type, a bright type, a darktype, a flue-cured type, an air-cured type, a fire-cured type, a Turkishtype, and an Oriental type.
 64. A tobacco product comprising cured plantmaterial from the Nicotiana tabacum plant of claim
 57. 65. The tobaccoproduct of claim 64, wherein the tobacco product is selected from thegroup consisting of a cigar, a cigarette, pipe tobacco, a cigarillo, anon-ventilated or vented recess filter cigarette, a dissolving strip, agum, a tablet, snuff, and chewing tobacco.
 66. A Nicotiana tabacum seedfrom the Nicotiana tabacum plant of claim 57, wherein the seed comprisesthe first mutation, the second mutation, and the third mutation.